Medium containing flt3 ligand for culturing hematophoietic cells

ABSTRACT

Ligands for flt3 receptors capable of transducing self-renewal signals to regulate the growth, proliferation or differentiation of progenitor cells and stem cells are disclosed. The invention is directed to flt3-L as an isolated protein, the DNA encoding the flt3-L, host cells transfected with cDNAs encoding flt3-L, compositions comprising flt3-L, methods of improving gene transfer to a mammal using flt3-L, and methods of improving transplantations using flt3-L. Flt3 -L finds use in treating patients with anemia, AIDS and various cancers.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser.No.08/444,627, filed May 19, 1995, now pending, which is a divisional ofU.S. application Ser. No, 08/243,545 filed May 11, 1994, now allowed andissued as U.S. Pat. No. 5,554,512, which is a continuation-in-part ofU.S. application Ser. No. 08/209,502 filed Mar. 7, 1994, now abandoned,which is a continuation-in-part of U.S. application Ser. No. 08/162,407,filed Dec. 3, 1993, now abandoned, which is a continuation-in-part ofU.S. application Ser. No. 08/111,758, filed Aug. 25, 1993, nowabandoned, which is a continuation-in-part of U.S. application Ser. No.08/106,463, filed Aug. 12, 1993, now abandoned, which is acontinuation-in-part of U.S. application Ser. No. 08/068,394 filed May24, 1993, now abandoned.

FIELD OF THE INVENTION

[0002] The present invention relates to mammalian flt3-ligands, thenucleic acids encoding such ligands, processes for production ofrecombinant flt3-ligands, pharmaceutical compositions containing suchligands, and their use in various therapies.

BACKGROUND OF THE INVENTION

[0003] Blood cells originate from hematopoietic stem cells that becomecommitted to differentiate along certain lineages, i.e., erythroid,megakaryocytic, granulocytic, monocytic, and lymphocytic. Cytokines thatstimulate the proliferation and maturation of cell precursors are calledcolony stimulating factors (“CSFs”). Several CSFs are produced byT-lymphocytes, including interleukin-3 (“IL-3”), granulocyte-monocyteCSF (GM-CSF), granulocyte CSF (G-CSF), and monocyte CSF (M-CSF). TheseCSFs affect both mature cells and stem cells. Heretofore no factors havebeen discovered that are able to predominantly affect stem cells.

[0004] Tyrosine kinase receptors (“TKRs”) are growth factor receptorsthat regulate the proliferation and differentiation of a number of cells(Yarden, Y. & Ullrich, A. Annu. Rev. Biochem., 57, 443-478, 1988; andCadena, D. L. & Gill, G. N. FASEB J., 6, 2332-2337, 1992). Certain TKRsfunction within the hematopoietic system. For example, signaling throughthe colony-stimulating factor type 1 (“CSF-1”), receptor c-fms regulatesthe survival, growth and differentiation of monocytes (Stanley et al.,J. Cell Biochem., 21, 151-159, 1983). Steel factor (“SF”, also known asmast cell growth factor, stem cell factor or kit ligand), acting throughc-kit, stimulates the proliferation of cells in both myeloid andlymphoid compartments.

[0005] Flt3 (Rosnet et al. Oncogene, 6, 1641-1650, 1991) and flk-2(Matthews et al., Cell, 65, 1143-1152, 1991) are variant forms of a TKRthat is related to the c-fms and c-kit receptors. The flk-2 gene productis expressed on hematopoietic and progenitor cells, while the flt3 geneproduct has a more general tissue distribution. The flt3 and flk-2receptor proteins are similar in amino acid sequence and vary at twoamino acid residues in the extracellular domain and diverge in a 31amino acid segment located near the C-termini (Lyman et al., Oncogene,8, 815-822, 1993).

[0006] Flt3-ligand (“flt3-L”) has been found to regulate the growth anddifferentiation of progenitor and stem cells and is likely to possessclinical utility in treating hematopoietic disorders, in particular,aplastic anemia and myelodysplastic syndromes. Additionally, flt3-L willbe useful in allogeneic, syngeneic or autologous bone marrow transplantsin patients undergoing cytoreductive therapies, as well as cellexpansion. Flt3-L will also be useful in gene therapy and progenitor andstem cell mobilization systems.

[0007] Cancer is treated with cytoreductive therapies that involveadministration of ionizing radiation or chemical toxins that killrapidly dividing cells. Side effects typically result from cytotoxiceffects upon normal cells and can limit the use of cytoreductivetherapies. A frequent side effect is myelosuppression, or damage to bonemarrow cells that give rise to white and red blood cells and platelets.As a result of myelosuppression, patients develop cytopenia, or bloodcell deficits, that increase risk of infection and bleeding disorders.

[0008] Cytopenias increase morbidity, mortality, and lead tounder-dosing in cancer treatment. Many clinical investigators havemanipulated cytoreductive therapy dosing regimens and schedules toincrease dosing for cancer therapy, while limiting damage to bonemarrow. One approach involves bone marrow or peripheral blood celltransplants in which bone marrow or circulating hematopoietic progenitoror stem cells are removed before cytoreductive therapy and thenreinfused following therapy to restore hematopoietic function. U.S. Pat.No. 5,199,942, incorporated herein by reference, describes a method forusing GM-CSF, IL-3, SF, GM-CSF/IL-3 fusion proteins, erythropoietin(“EPO”) and combinations thereof in autologous transplantation regimens.

[0009] High-dose chemotherapy is therapeutically beneficial because itcan produce an increased frequency of objective response in patientswith metastatic cancers, particularly breast cancer, when compared tostandard dose therapy. This can result in extended disease-freeremission for some even poor-prognosis patients. Nevertheless, high-dosechemotherapy is toxic and many resulting clinical complications arerelated to infections, bleeding disorders and other effects associatedwith prolonged periods of myelosuppression.

[0010] Myelodysplastic syndromes are stem cell disorders characterizedby impaired cellular maturation, progressive pancytopenia, andfunctional abnormalities of mature cells. They have also beencharacterized by variable degrees of cytopenia, ineffectiveerythropoiesis and myelopoiesis with bone marrow cells that are normalor increased in number and that have peculiar morphology. Bennett et.al. (Br. J. Haematol. 1982; 51:189-199) divided these disorders intofive subtypes: refractory anemia, refractory anemia with ringedsideroblasts, refractory anemia with excess blasts, refractory anemiawith excess blasts in transformation, and chronic myelomonocyticleukemia. Although a significant percentage of these patients developacute leukemia, a majority die from infectious or hemorrhagiccomplications. Treatment of theses syndromes with retinoids, vitamin D,and cytarabine has not been successful. Most of the patients sufferingfrom these syndromes are elderly and are not suitable candidates forbone marrow transplantation or aggressive antileukemic chemotherapy.

[0011] Aplastic anemia is another disease entity that is characterizedby bone marrow failure and severe pancytopenia. Unlike myelodysplasticsyndrome, the bone marrow is acellular or hypocellular in this disorder.Current treatments include bone marrow transplantation from ahistocompatible donor or immunosuppressive treatment with antithymocyteglobulin (ATG). Similarly to myelodysplastic syndrome, most patientssuffering from this syndrome are elderly and are unsuitable for bonemarrow transplantation or for aggressive antileukemic chemotherapy.Mortality in these patients is exceedingly high from infectious orhemorrhagic complications.

[0012] Anemia is common in patients with acquired immune deficiencysyndrome (AIDS). The anemia is usually more severe in patients receivingzidovudine therapy. Many important retroviral agents, anti-infectives,and anti-neoplastics suppress erythropoiesis. Recombinant EPO has beenshown to normalize the patient's hematocrit and hemaoglobin levels,however, usually very high doses are required. A growth factor thatstimulates proliferation of the erythroid lineage could be used alone orin combination with EPO or other growth factors to treat such patientsand reduce the number of transfusions required. A growth factor thatcould also increase the number of T cells would find particular use intreating AIDS patients.

SUMMARY OF THE INVENTION

[0013] The present invention pertains to biologically active flt3-ligand(flt3-L) as an isolated or homogeneous protein. In addition, theinvention is directed to isolated DNAs encoding flt3-L and to expressionvectors comprising a cDNA encoding flt3-L. Within the scope of thisinvention are host cells that have been transfected or transformed withexpression vectors that comprise a cDNA encoding flt3-L, and processesfor producing flt3-L by culturing such host cells under conditionsconducive to expression of flt3-L.

[0014] Flt3-L can be used to prepare pharmaceutical compositions to beused in allogeneic, syngeneic or autologous transplantation methods.Pharmaceutical compo- sitions may comprise flt3-L alone or incombination with other growth factors, such as interleukins, colonystimulating factors, protein tyrosine kinases and cytokines.

[0015] The invention includes methods of using flt3-L compositions ingene therapy and in treatment of patients suffering from myelodysplasticsyndrome, aplastic anemia, HIV infection (AIDS) and cancers, such asbreast cancer, lymphoma, small cell lung cancer, multiple myeloma,neuroblastoma, acute leukemia, testicular tumors, and ovarian cancer.

[0016] The present invention also pertains to antibodies, and inparticular monoclonal antibodies, that are immunoreactive with flt3-L.Fusion proteins comprising a soluble portion of flt3-L and the constantdomain of an immunoglobulin protein are also embodied in the invention.

[0017] The present invention also is directed to the use of flt3-L inperipheral blood progenitor or stem cell transplanation procedures.Typically, peripheral blood progenitor cells or stem cells are removedfrom a patient prior to myelosuppressive cytoreductive therapy, and thenreadministered to the patient concurrent with or following cytoreductivetherapy to counteract the myelosuppressive effects of such therapy. Thepresent invention provides for the use of an effective amount of flt3-Lin at least one of the following manners: (i) flt3-L is administered tothe patient prior to collection of the progenitor or stem cells toincrease or mobilize the numbers of such circulating cells; (ii)following collection of the patient's progenitor or stem cells, flt3-Lis used to expand such cells ex vivo; and (iii) flt3-L is administeredto the patient following transplantation of the collected progenitor orstem cells to facilitate engraftment thereof. The transplantation methodof the invention can further comprise the use of an effective amount ofa cytokine in sequential or concurrent combination with the flt3-L. Suchcytokines include, but are not limited to interleukins (“IL”) IL-1,IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12,IL-13, IL-14 or IL-15, a CSF selected from the group consisting ofG-CSF, GM-CSF, M-CSF, or GM-CSF/IL-3 fusions, or other growth factorssuch as CSF-1, SF, EPO, leukemia inhibitory factor (“LIF”) or fibroblastgrowth factor (“FGF”). The flt3-L is also useful in the same way forsyngeneic or allogeneic transplantations.

[0018] The invention further includes a progenitor or stem cellexpansion media comprising cell growth media, autologous serum, andflt3-L alone or in combination with a cytokine from the group listedabove.

[0019] The invention further includes the use of flt3-L to expandprogenitor or stem cells collected from umbilical cord blood. Theexpansion may be performed with flt3-L alone or in sequential orconcurrent combination with a cytokine from the group listed above.

[0020] The invention further includes the use of flt3-L in gene therapy.Flt3-L permits proliferation and culturing of the early hematopoieticprogenitor or stem cells that are to be transfected with exogenous DNAfor use in gene therapy. Alternatively, a cDNA encoding flt3-L may betransfected into cells in order to ultimately deliver its gene productto the targeted cell or tissue.

[0021] In addition, the invention includes the use of flt3-L tostimulate production of erythroid cells in vivo for the treatment ofanemia. Such use comprises administering flt3-L to the patient in needof such erythroid cell stimulation in conjunction with or followingcytoreductive therapy. The treatment can include co-administration ofanother growth factor selected from the cytokines from the group listedabove. Preferred cytokines for use in this treatment include EPO, IL-3,G-CSF and GM-CSF. Such treatment is especially useful for AIDS patients,and preferably for AIDS patients receiving AZT therapy.

[0022] Since flt3-L stimulates the production of stem cells, othernon-hematopoietic stem cells bearing flt3 receptors can be affected bythe flt3-L of the invention. Flt3-L is useful in in vitro fertilizationprocedures and can be used in vivo in the treatment of infertilityconditions. In the gut, the flt3 ligand is useful in treating intestinaldamage resulting from irradiation or chemotherapy. The flt3-L can bealso used to treat patients infected with the human immunodeficiencyvirus (HIV). Such treatment would encompass the administration of theflt3-L to stimulate in vivo production, as well as the ex vivoexpansion, of T cells and erythroid cells. Such treatment can preventthe deficiency of T cells, in particular CD4-positive T cells, and mayelevate the patient's immune reponse against the virus, therebyimproving the quality of life of the patient. The flt3-L can be used tostimulate the stem cells that lead to the development of hair follicles,thereby promoting hair growth.

[0023] In addition, flt3-L can be bound to a solid phase matrix and usedto affinity-purify or separate cells that express flt3 on their cellsurface. The invention encompasses separating cells having the flt3receptor on the surface thereof from a mixture of cells in solution,comprising contacting the cells in the mixture with a contacting surfacehaving a flt3-binding molecule thereon, and separating the contactingsurface and the solution. Once separated, the cells can be expanded exvivo using flt3-L and administered to a patient.

DETAILED DESCRIPTION OF THE INVENTION

[0024] A cDNA encoding murine flt3-L has been isolated and is disclosedin SEQ ID NO: 1. A cDNA encoding human flt3-L also has been isolated andis disclosed in SEQ ID NO: 5. This discovery of cDNAs encoding murineand human flt3-L enables construction of expression vectors comprisingcDNAs encoding flt3-L; host cells transfected or transformed with theexpression vectors; biologically active murine and human flt3-L ashomogeneous proteins; and antibodies immunoreactive with the murine andthe human flt3-L.

[0025] Flt3-L is useful in the enhancement, stimulation, proliferationor growth of cells expressing the flt3 receptor, includingnon-hematopoietic cells. Since the flt3 receptor is found in the brain,placenta, and tissues of nervous and hematopoietic origin, and findsdistribution in the testis, ovaries, lymph node, spleen, thymus andfetal liver, treatment of a variety of conditions associated with tissuedamage thereof is possible. While not limited to such, particular usesof the flt3-L are described infra.

[0026] As used herein, the term “flt3-L” refers to a genus ofpolypeptides that bind and complex independently with flt3 receptorfound on progenitor and stem cells. The term “flt3-L” encompassesproteins having the amino acid sequence 1 to 231 of SEQ ID NO: 2 or theamino acid sequence 1 to 235 of SEQ ID NO: 6, as well as those proteinshaving a high degree of similarity or a high degree of identity with theamino acid sequence 1 to 231 of SEQ ID NO: 2 or the amino acid sequence1 to 235 of SEQ ID NO: 6, and which proteins are biologically active andbind the flt3 receptor. In addition, the term refers to biologicallyactive gene products of the DNA of SEQ ID NO: 1 or SEQ ID NO: 5. Furtherencompassed by the term “flt3-L” are the membrane-bound proteins (whichinclude an intracellular region, a membrane region, and an extracellularregion), and soluble or truncated proteins which comprise primarily theextracellular portion of the protein, retain biological activity and arecapable of being secreted. Specific examples of such soluble proteinsare those comprising the sequence of amino acids 28-163 of SEQ ID NO: 2and amino acids 28-160 of SEQ ID NO: 6.

[0027] The term “biologically active” as it refers to flt3-L, means thatthe flt3-L is capable of binding to flt3. Alternatively, “biologicallyactive” means the flt3-L is capable of transducing a stimulatory signalto the cell through the membrane-bound flt3.

[0028] “Isolated” means that flt3-L is free of association with otherproteins or polypeptides, for example, as a purification product ofrecombinant host cell culture or as a purified extract.

[0029] A “flt3-L variant” as referred to herein, means a polypeptidesubstantially homologous to native flt3-L, but which has an amino acidsequence different from that of native flt3-L (human, murine or othermammalian species) because of one or more deletions, insertions orsubstitutions. The variant amino acid sequence preferably is at least80% identical to a native flt3-L amino acid sequence, most preferably atleast 90% identical. The percent identity may be determined, forexample, by comparing sequence information using the GAP computerprogram, version 6.0 described by Devereux et al. (Nucl. Acids Res.12:387, 1984) and available from the University of Wisconsin GeneticsComputer Group (UWGCG). The GAP program utilizes the alignment method ofNeedleman and Wunsch (J. Mol. Biol. 48:443, 1970), as revised by Smithand Waterman (Adv. Appl. Math 2:482, 1981). The preferred defaultparameters for the GAP program include: (1) a unary comparison matrix(containing a value of 1 for identities and 0 for non-identities) fornucleotides, and the weighted comparison matrix of Gribskov and Burgess,Nucl. Acids Res. 14:6745, 1986, as described by Schwartz and Dayhoff,eds., Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, pp. 353-358, 1979; (2) a penalty of 3.0 for eachgap and an additional 0.10 penalty for each symbol in each gap; and (3)no penalty for end gaps. Variants may comprise conservativelysubstituted sequences, meaning that a given amino acid residue isreplaced by a residue having similar physiochemical characteristics.Examples of conservative substitutions include substitution of onealiphatic residue for another, such as Ile, Val, Leu, or Ala for oneanother, or substitutions of one polar residue for another, such asbetween Lys and Arg; Glu and Asp; or Gln and Asn. Other suchconservative substitutions, for example, substitutions of entire regionshaving similar hydrophobicity characteristics, are well known. Naturallyoccurring flt3-L variants are also encompassed by the invention.Examples of such variants are proteins that result from alternate mRNAsplicing events or from proteolytic cleavage of the flt3-L protein,wherein the flt3-L binding property is retained. Alternate splicing ofMRNA may yield a truncated but biologically active flt3-L protein, suchas a naturally occurring soluble form of the protein, for example.Variations attributable to proteolysis include, for example, differencesin the N- or C-termini upon expression in different types of host cells,due to proteolytic removal of one or more terminal amino acids from theflt3-L protein (generally from 1-5 terminal amino acids).

[0030] The term “autologous transplantation” is described in U.S. Pat.No. 5,199,942, which is incorporated herein by reference. Briefly, theterm means a method for conducting autologous hematopoietic progenitoror stem cell transplantation, comprising: (1) collecting hematopoieticprogenitor cells or stem cells from a patient prior to cytoreductivetherapy; (2) expanding the hematopoietic progenitor cells or stem cellsex vivo with flt3-L to provide a cellular preparation comprisingincreased numbers of hematopoietic progenitor cells or stem cells; and(3) administering the cellular preparation to the patient in conjunctionwith or following cytoreductive therapy. Progenitor and stem cells maybe obtained from peripheral blood harvest or bone marrow explants.Optionally, one or more cytokines, selected from the group listed abovecan be combined with flt3-L to aid in the proliferation of particularhematopoietic cell types or affect the cellular function of theresulting proliferated hematopoietic cell population. Of the foregoing,SF, IL-1, IL-3, EPO, G-CSF, GM-CSF and GM-CSF/IL-3 fusions arepreferred, with G-CSF, GM-CSF and GM-CSF/IL-3 fusions being especiallypreferred. The term “allogeneic transplantation” means a method in whichbone marrow or peripheral blood progenitor cells or stem cells areremoved from a mammal and administered to a different mammal of the samespecies. The term “syngeneic transplantation” means the bone marrowtransplantation between gentically identical mammals.

[0031] The transplantation method of the invention described aboveoptionally comprises a preliminary in vivo procedure comprisingadministering flt3-L alone or in sequential or concurrent combinationwith a recruitment growth factor to a patient to recruit thehematopoietic cells into peripheral blood prior to their harvest.Suitable recruitment factors are listed above, and preferred recruitmentfactors are flt3-L, SF, IL-1 and IL-3.

[0032] The method of the invention described above optionally comprisesa subsequent in vivo procedure comprising administering flt3-L alone orin sequential or concurrent combination with an engraftment growthfactor to a patient following transplantation of the cellularpreparation to facilitate engraftment and augment proliferation ofengrafted hematopoietic progenitor or stem cells from the cellularpreparation. Suitable engraftment factors are listed above, and thepreferred engraftment factors are GM-CSF, G-CSF, IL-3, IL-1, EPO andGM-CSF/IL-3 fusions.

[0033] The invention further includes a progenitor or stem cellexpansion media comprising cell growth media, autologous serum, andflt3-L alone or in combination with a cytokine growth factor from thelist above. Preferred growth factors are SF, GM-CSF, IL-3, IL-1, G-CSF,EPO, and GM-CSF/IL-3 fusions.

[0034] In particular, flt3-L can be used to stimulate the proliferationof hematopoietic and non-hematopoietic stem cells. Such stimulation isbeneficial when specific tissue damage has occurred to these tissues. Assuch, flt3-L may be useful in treating neurological damage and may be agrowth factor for nerve cells. It is probable that flt3-L would beuseful in in vitro fertilization procedures and likely can be used invivo in the treatment of infertility conditions. Flt3-L would be usefulin treating intestinal damage resulting from irradiation orchemotherapy. Since the flt3 receptor is distributed on stem cellsleading to the development of hair follicles, flt3-L would likely beuseful to promote hair growth.

[0035] Since flt3-L has been shown to stimulate T cell proliferation aswell as erythrocytes (see Examples, infra), flt3-L finds use in thetreatment of patients infected with the human immunodeficiency virus(HIV). Such treatment would encompass the administration of flt3-L tostimulate in vivo production, as well as the ex vivo expansion, of Tcells. In addition, flt3-L can prevent the deficiency of CD4⁺ T cells.Such treatment may elevate or maintain a patient's immune reponseagainst the virus, thereby improving or maintaining a patient's qualityof life. In addition, such in vivo treatment would stimulate cells ofthe erythroid lineage, thereby improving a patient's hematocrit andhemaglobin levels. Flt3-L can be administered in this setting alone orin sequential or concurrent combination with cytokines selected from thegroup listed above.

[0036] Flt3-L is useful in gene therapy due to its specificity forprogenitor and stem cells. Gene therapy involves administration ofexogenous DNA-transfected cells to a host that are allowed to engraft.See e.g., Boggs, International J. Cell Cloning, 8:80-96, (1990); Kohnet. al., Cancer Invest., 7(2):179-192 (1989); Lehn, Bone MarrowTranspl., 5:287-293 (1990); and Verma, Scientific American, pp. 68-84(1990). Using gene therapy methods known in the art, a method oftransferring a gene to a mammal comprises the steps of (a) culturingearly hematopoietic cells in media comprising flt3-L alone or insequential or concurrent combination with a cyokine selected from thegroup listed above; (b) transfecting the cultured cells from step (a)with the exogenous gene; and (c) administering the transfected cells tothe mammal. Within this method is the novel method of transfectingprogenitor or stem cells with a gene comprising the steps of: (a) and(b) above. Furthermore, using the same or simolar methods, the cDNAencoding the flt3-L can be transfected into such delivery cells todeliver the flt3-L gene product to the targetted tissue.

[0037] Example 1 describes the construction of a novel flt3:Fc fusionprotein utilized in the screening for flt3-L. Other antibody Fc regionsmay be substituted for the human IgG1 Fc region described in Example 1.Other suitable Fc regions are those that can bind with high affinity toprotein A or protein G, and include the Fc region of human IgG1 orfragments of the human or murine IgG1 Fc region, e.g., fragmentscomprising at least the hinge region so that interchain disulfide bondswill form. The flt3:Fc fusion protein offers the advantage of beingeasily purified. In addition, disulfide bonds form between the Fcregions of two separate fusion protein chains, creating dimers. Thedimeric flt3:Fc receptor was chosen for the potential advantage ofhigher affinity binding of flt3-L, in view of the possibility that theligand being sought would be multimeric.

[0038] As described supra., an aspect of the invention is soluble flt3-Lpolypeptides. Soluble flt3-L polypeptides comprise all or part of theextracellular domain of a native flt3-L but lack the transmembraneregion that would cause retention of the polypeptide on a cell membrane.Soluble flt3-L polypeptides advantageously comprise the native (or aheterologous) signal peptide when initially synthesized to promotesecretion, but the signal peptide is cleaved upon secretion of flt3-Lfrom the cell. Soluble flt3-L polypeptides encompassed by the inventionretain the ability to bind the flt3 receptor. Indeed, soluble flt3-L mayalso include part of the transmembrane region or part of the cytoplasmicdomain or other sequences, provided that the soluble flt3-L protein canbe secreted.

[0039] Soluble flt3-L may be identified (and distinguished from itsnon-soluble membrane-bound counterparts) by separating intact cellswhich express the desired protein from the culture medium, e.g., bycentrifugation, and assaying the medium (supernatant) for the presenceof the desired protein. The presence of flt3-L in the medium indicatesthat the protein was secreted from the cells and thus is a soluble formof the desired protein.

[0040] Soluble forms of flt3-L possess many advantages over the nativebound flt3-L protein. Purification of the proteins from recombinant hostcells is feasible, since the soluble proteins are secreted from thecells. Further, soluble proteins are generally more suitable forintravenous administration.

[0041] Examples of soluble flt3-L polypeptides include those comprisinga substantial portion of the extracellular domain of a native flt3-Lprotein. Such soluble mammalian flt3-L proteins comprise amino acids 28through 188 of SEQ ID NO: 2 or amino acids 28 through 182 of SEQ ID NO:6. In addition, truncated soluble flt3-L proteins comprising less thanthe entire extracellular domain are included in the invention. Suchtruncated soluble proteins are represented by the sequence of aminoacids 28-163 of SEQ ID NO: 2, and amino acids 28-160 of SEQ ID NO: 6.When initially expressed within a host cell, soluble flt3-L mayadditionally comprise one of the heterologous signal peptides describedbelow that is functional within the host cells employed. Alternatively,the protein may comprise the native signal peptide, such that themammalian flt3-L comprises amino acids 1 through 188 of SEQ ID NO: 2 oramino acids 1 through 182 of SEQ ID NO: 6. In one embodiment of theinvention, soluble flt3-L was expressed as a fusion protein comprising(from N- to C-terminus) the yeast α factor signal peptide, a FLAG®peptide described below and in U.S. Pat. No. 5,011,912, and solubleflt3-L consisting of amino acids 28 to 188 of SEQ ID NO: 2. Thisrecombinant fusion protein is expressed in and secreted from yeastcells. The FLAG® peptide facilitates purification of the protein, andsubsequently may be cleaved from the soluble flt3-L using bovine mucosalenterokinase. Isolated DNA sequences encoding soluble flt3-L proteinsare encompassed by the invention.

[0042] Truncated flt3-L, including soluble polypeptides, may be preparedby any of a number of conventional techniques. A desired DNA sequencemay be chemically synthesized using techniques known per se. DNAfragments also may be produced by restriction endonuclease digestion ofa full length cloned DNA sequence, and isolated by electrophoresis onagarose gels. Linkers containing restriction endonuclease cleavagesite(s) may be employed to insert the desired DNA fragment into anexpression vector, or the fragment may be digested at cleavage sitesnaturally present therein. The well known polymerase chain reactionprocedure also may be employed to amplify a DNA sequence encoding adesired protein fragment. As a further alternative, known mutagenesistechniques may be employed to insert a stop codon at a desired point,e.g., immediately downstream of the codon for the last amino acid of theextracellular domain.

[0043] In another approach, enzymatic treatment (e.g., using Bal 31exonuclease) may be employed to delete terminal nucleotides from a DNAfragment to obtain a fragment having a particular desired terminus.Among the commercially available linkers are those that can be ligatedto the blunt ends produced by Bal 31 digestion, and which containrestriction endonuclease cleavage site(s). Alternatively,oligonucleotides that reconstruct the N- or C-terminus of a DNA fragmentto a desired point may be synthesized and ligated to the DNA fragment.The synthesized oligonucleotide may contain a restriction endonucleasecleavage site upstream of the desired coding sequence and position aninitiation codon (ATG) at the N-terminus of the coding sequence.

[0044] As stated above, the invention provides isolated or homogeneousflt3-L polypeptides, both recombinant and non-recombinant. Variants andderivatives of native flt3-L proteins that retain the desired biologicalactivity (e.g., the ability to bind flt3) may be obtained by mutationsof nucleotide sequences coding for native flt3-L polypeptides.Alterations of the native amino acid sequence may be accomplished by anyof a number of conventional methods. Mutations can be introduced atparticular loci by synthesizing oligonucleotides containing a mutantsequence, flanked by restriction sites enabling ligation to fragments ofthe native sequence. Following ligation, the resulting reconstructedsequence encodes an analog having the desired amino acid insertion,substitution, or deletion.

[0045] Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures can be employed to provide an altered gene whereinpredetermined codons can be altered by substitution, deletion orinsertion. Exemplary methods of making the alterations set forth aboveare disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al.(Genetic Engineering: Principles and Methods, Plenum Press, 1981);Kunkel (Proc. Natl. Acad. Sci. USA 82:488, 1985); Kunkel et al. (Methodsin Enzymol. 154:367, 1987); and U.S. Pat. Nos. 4,518,584 and 4,737,462all of which are incorporated by reference.

[0046] Flt3-L may be modified to create flt3-L derivatives by formingcovalent or aggregative conjugates with other chemical moieties, such asglycosyl groups, lipids, phosphate, acetyl groups and the like. Covalentderivatives of flt3-L may be prepared by linking the chemical moietiesto functional groups on flt3-L amino acid side chains or at theN-terminus or C-terminus of a flt3-L polypeptide or the extracellulardomain thereof. Other derivatives of flt3-L within the scope of thisinvention include covalent or aggregative conjugates of flt3-L or itsfragments with other proteins or polypeptides, such as by synthesis inrecombinant culture as N-terminal or C-terminal fusions. For example,the conjugate may comprise a signal or leader polypeptide sequence (e.g.the a-factor leader of Saccharomyces) at the N-terminus of a flt3-Lpolypeptide. The signal or leader peptide co-translationally orpost-translationally directs transfer of the conjugate from its site ofsynthesis to a site inside or outside of the cell membrane or cell wall.

[0047] Flt3-L polypeptide fusions can comprise peptides added tofacilitate purification and identification of flt3-L. Such peptidesinclude, for example, poly-His or the antigenic identification peptidesdescribed in U.S. Pat. No. 5,011,912 and in Hopp et al., Bio/Technology6:1204, 1988.

[0048] The invention further includes flt3-L polypeptides with orwithout associated native-pattern glycosylation. Flt3-L expressed inyeast or mammalian expression systems (e.g., COS-7 cells) may be similarto or significantly different from a native flt3-L polypeptide inmolecular weight and glycosylation pattern, depending upon the choice ofexpression system. Expression of flt3-L polypeptides in bacterialexpression systems, such as E. coli, provides non-glycosylatedmolecules.

[0049] Equivalent DNA constructs that encode various additions orsubstitutions of amino acid residues or sequences, or deletions ofterminal or internal residues or sequences not needed for biologicalactivity or binding are encompassed by the invention. For example,N-glycosylation sites in the flt3-L extracellular domain can be modifiedto preclude glycosylation, allowing expression of a reduced carbohydrateanalog in mammalian and yeast expression systems. N-glycosylation sitesin eukaryotic polypeptides are characterized by an amino acid tripletAsn-X-Y, wherein X is any amino acid except Pro and Y is Ser or Thr. Themurine and human flt3-L proteins each comprise two such triplets, atamino acids 127-129 and 152-154 of SEQ ID NO: 2, and at amino acids126-128 and 150-152 of SEQ ID NO: 6, respectively. Appropriatesubstitutions, additions or deletions to the nucleotide sequenceencoding these triplets will result in prevention of attachment ofcarbohydrate residues at the Asn side chain. Alteration of a singlenucleotide, chosen so that Asn is replaced by a different amino acid,for example, is sufficient to inactivate an N-glycosylation site. Knownprocedures for inactivating N-glycosylation sites in proteins includethose described in U.S. Pat. No. 5,071,972 and EP 276,846, herebyincorporated by reference.

[0050] In another example, sequences encoding Cys residues that are notessential for biological activity can be altered to cause the Cysresidues to be deleted or replaced with other amino acids, preventingformation of incorrect intramolecular disulfide bridges uponrenaturation. Other equivalents are prepared by modification of adjacentdibasic amino acid residues to enhance expression in yeast systems inwhich KEX2 protease activity is present. EP 212,914 discloses the use ofsite-specific mutagenesis to inactivate KEX2 protease processing sitesin a protein. KEX2 protease processing sites are inactivated bydeleting, adding or substituting residues to alter Arg-Arg, Arg-Lys, andLys-Arg pairs to eliminate the occurrence of these adjacent basicresidues. Lys-Lys pairings are considerably less susceptible to KEX2cleavage, and conversion of Arg-Lys or Lys-Arg to Lys-Lys represents aconservative and preferred approach to inactivating KEX2 sites. Bothmurine and human flt3-L contain two KEX2 protease processing sites atamino acids 216-217 and 217-218 of SEQ ID NO: 2 and at amino acids211-212 and 212-213 of SEQ ID NO: 6, respectively.

[0051] Nucleic acid sequences within the scope of the invention includeisolated DNA and RNA sequences that hybridize to the native flt3-Lnucleotide sequences disclosed herein under conditions of moderate orsevere stringency, and which encode biologically active flt3-L.Conditions of moderate stringency, as defined by Sambrook et al.Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1, pp. 1.101-104,Cold Spring Harbor Laboratory Press, (1989), include use of a prewashingsolution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) and hybridizationconditions of about 55° C., 5×SSC, overnight. Conditions of severestringency include higher temperatures of hybridization and washing. Theskilled artisan will recognize that the temperature and wash solutionsalt concentration may be adjusted as necessary according to factorssuch as the length of the probe.

[0052] Due to the known degeneracy of the genetic code wherein more thanone codon can encode the same amino acid, a DNA sequence may vary fromthat shown in SEQ ID NO: 1 and SEQ ID NO: 5 and still encode an flt3-Lprotein having the amino acid sequence of SEQ ID NO: 2 and SEQ ID NO: 6,respectively. Such variant DNA sequences may result from silentmutations (e.g., occurring during PCR amplification), or may be theproduct of deliberate mutagenesis of a native sequence.

[0053] The invention provides equivalent isolated DNA sequences encodingbiologically active flt3-L, selected from: (a) DNA derived from thecoding region of a native mammalian flt3-L gene; (b) cDNA comprising thenucleotide sequence presented in SEQ ID NO: 1 or SEQ ID NO: 5; (c) DNAcapable of hybridization to a DNA of (a) under moderately stringentconditions and which encodes biologically active flt3-L; and (d) DNAwhich is degenerate as a result of the genetic code to a DNA defined in(a), (b) or (c) and which encodes biologically active flt3-L. Flt3-Lproteins encoded by such DNA equivalent sequences are encompassed by theinvention.

[0054] DNA that are equivalents to the DNA sequence of SEQ ID NO: 1 orSEQ ID NO: 5, will hybridize under moderately stringent conditions tothe native DNA sequence that encode polypeptides comprising amino acidsequences of 28-163 of SEQ ID NO: 2 or 28-160 of SEQ ID NO: 6. Examplesof flt3-L proteins encoded by such DNA, include, but are not limited to,flt3-L fragments (soluble or membrane-bound) and flt3-L proteinscomprising inactivated N-glycosylation site(s), inactivated KEX2protease processing site(s), or conservative amino acid substitution(s),as described above. Flt3-L proteins encoded by DNA derived from othermammalian species, wherein the DNA will hybridize to the cDNA of SEQ IDNO: 1 or SEQ ID NO: 5, are also encompassed.

[0055] Variants possessing the requisite ability to bind flt3 receptormay be identified by any suitable assay. Biological activity of flt3-Lmay be determined, for example, by competition for binding to the ligandbinding domain of flt3 receptor (i.e. competitive binding assays).

[0056] One type of a competitive binding assay for a flt3-L polypeptideuses a radiolabeled, soluble human flt3-L and intact cells expressingcell surface flt3 receptors. Instead of intact cells, one couldsubstitute soluble flt3 receptors (such as a flt3:Fc fusion protein)bound to a solid phase through the interaction of a Protein A, Protein Gor an antibody to the flt3 or Fc portions of the molecule, with the Fcregion of the fusion protein. Another type of competitive binding assayutilizes radiolabeled soluble flt3 receptors such as a flt3:Fc fusionprotein, and intact cells expressing flt3-L. Alternatively, solubleflt3-L could be bound to a solid phase to positively select flt3expressing cells.

[0057] Competitive binding assays can be performed followingconventional methodology. For example, radiolabeled flt3-L can be usedto compete with a putative flt3-L homolog to assay for binding activityagainst surface-bound flt3 receptors. Qualitative results can beobtained by competitive autoradiographic plate binding assays, orScatchard plots may be utilized to generate quantitative results.

[0058] Alternatively, flt3-binding proteins, such as flt3-L andanti-flt3 antibodies, can be bound to a solid phase such as a columnchromatography matrix or a similar substrate suitable for identifying,separating or purifying cells that express the flt3 receptor on theirsurface. Binding of flt3-binding proteins to a solid phase contactingsurface can be accomplished by any means, for example, by constructing aflt3-L:Fc fusion protein and binding such to the solid phase through theinteraction of Protein A or Protein G. Various other means for fixingproteins to a solid phase are well known in the art and are suitable foruse in the present invention. For example, magnetic microspheres can becoated with flt3 -binding proteins and held in the incubation vesselthrough a magnetic field. Suspensions of cell mixtures containinghematopoietic progenitor or stem cells are contacted with the solidphase that has flt3-binding proteins thereon. Cells having the flt3receptor on their surface bind to the fixed flt3-binding protein andunbound cells then are washed away. This affinity-binding method isuseful for purifying, screening or separating such flt3-expressing cellsfrom solution. Methods of releasing positively selected cells from thesolid phase are known in the art and encompass, for example, the use ofenzymes. Such enzymes are preferably non-toxic and non-injurious to thecells and are preferably directed to cleaving the cell-surface bindingpartner. In the case of flt3:flt3-L interactions, the enzyme preferablywould cleave the flt3 receptor, thereby freeing the resulting cellsuspension from the “foreign” flt3-L material. The purified cellpopulation then may be expanded ex vivo prior to transplantation to apatient in an amount sufficient to reconstitute the patient'shematopoietic and immune system.

[0059] Alternatively, mixtures of cells suspected of containing flt3⁺cells first can be incubated with a biotinylated flt3-binding protein.Incubation periods are typically at least one hour in duration to ensuresufficient binding to flt3. The resulting mixture then is passed througha column packed with avidin-coated beads, whereby the high affinity ofbiotin for avidin provides the binding of the cell to the beads. Use ofavidin-coated beads is known in the art. See Berenson, et al. J. Cell.Biochem., 10D:239 (1986). Wash of unbound material and the release ofthe bound cells is performed using conventional methods.

[0060] In the methods described above, suitable flt3-binding proteinsare flt3-L, anti-flt3 antibodies, and other proteins that are capable ofhigh-affinity binding of flt3. A preferred flt3-binding protein isflt3-L.

[0061] As described above, flt3-L of the invention can be used toseparate cells expressing flt3 receptors. In an alternative method,flt3-L or an extracellular domain or a fragment thereof can beconjugated to a detectable moiety such as ¹²⁵I to detect flt3 expressingcells. Radiolabeling with ¹²⁵I can be performed by any of severalstandard methodologies that yield a functional ¹²⁵I-flt3-L moleculelabeled to high specific activity. Or an iodinated or biotinylatedantibody against the flt3 region or the Fc region of the molecule couldbe used. Another detectable moiety such as an enzyme that can catalyze acolorimetric or fluorometric reaction, biotin or avidin may be used.Cells to be tested for flt3 receptor expression can be contacted withlabeled flt3-L. After incubation, unbound labeled flt3-L is removed andbinding is measured using the detectable moiety.

[0062] The binding characteristics of flt3-L (including variants) mayalso be determined using the conjugated, soluble flt3 receptors (forexample, ¹²⁵I-flt3:Fc) in competition assays similar to those describedabove. In this case, however, intact cells expressing flt3 receptors, orsoluble flt3 receptors bound to a solid substrate, are used to measurethe extent to which a sample containing a putative flt3-L variantcompetes for binding with a conjugated a soluble flt3 to flt3-L.

[0063] Other means of assaying for flt3-L include the use of anti-flt3-Lantibodies, cell lines that proliferate in response to flt3-L, orrecombinant cell lines that express flt3 receptor and proliferate in thepresenvce of flt3-L. For example, the BAF/BO3 cell line lacks the flt3receptor and is IL-3 dependent. (See Hatakeyama, et al., Cell,59:837-845 (1989)). BAF/BO3 cells transfected with an expression vectorcomprising the flt3 receptor gene proliferate in response to either IL-3or flt3-L. An example of a suitable expression vector for transfectionof flt3 is the pCAV/NOT plasmid, see Mosley et al., Cell, 59:335-348(1989).

[0064] Flt3-L polypeptides may exist as oligomers, such ascovalently-linked or non-covalently-linked dimers or trimers. Oligomersmay be linked by disulfide bonds formed between cysteine residues ondifferent flt3-L polypeptides. In one embodiment of the invention, aflt3-L dimer is created by fusing flt3-L to the Fc region of an antibody(e.g., IgG1) in a manner that does not interfere with binding of flt3-Lto the flt3-ligand-binding domain. The Fc polypeptide preferably isfused to the C-terminus of a soluble flt3-L (comprising only theextracellular domain). General preparation of fusion proteins comprisingheterologous polypeptides fused to various portions of antibody-derivedpolypeptides (including the Fc domain) has been described, e.g., byAshkenazi et al. (PNAS USA 88:10535, 1991) and Byrn et al. (Nature344:677, 1990), hereby incorporated by reference. A gene fusion encodingthe flt3-L:Fc fusion protein is inserted into an appropriate expressionvector. Flt3-L:Fc fusion proteins are allowed to assemble much likeantibody molecules, whereupon interchain disulfide bonds form between Fcpolypeptides, yielding divalent flt3-L. If fusion proteins are made withboth heavy and light chains of an antibody, it is possible to form aflt3-L oligomer with as many as four flt3-L extracellular regions.Alternatively, one can link two soluble flt3-L domains with a peptidelinker.

[0065] Recombinant expression vectors containing a DNA encoding flt3-Lcan be prepared using well known methods. The expression vectors includea flt3-L DNA sequence operably linked to suitable transcriptional ortranslational regulatory nucleotide sequences, such as those derivedfrom a mammalian, microbial, viral, or insect gene. Examples ofregulatory sequences include transcriptional promoters, operators, orenhancers, an mRNA ribosomal binding site, and appropriate sequenceswhich control transcription and translation initiation and termination.Nucleotide sequences are “operably linked” when the regulatory sequencefunctionally relates to the flt3-L DNA sequence. Thus, a promoternucleotide sequence is operably linked to a flt3-L DNA sequence if thepromoter nucleotide sequence controls the transcription of the flt3-LDNA sequence. The ability to replicate in the desired host cells,usually conferred by an origin of replication, and a selection gene bywhich transformants are identified, may additionally be incorporatedinto the expression vector.

[0066] In addition, sequences encoding appropriate signal peptides thatare not naturally associated with flt3-L can be incorporated intoexpression vectors. For example, a DNA sequence for a signal peptide(secretory leader) may be fused in-frame to the flt3-L sequence so thatflt3-L is initially translated as a fusion protein comprising the signalpeptide. A signal peptide that is functional in the intended host cellsenhances extracellular secretion of the flt3-L polypeptide. The signalpeptide may be cleaved from the flt3-L polypeptide upon secretion offlt3-L from the cell.

[0067] Suitable host cells for expression of flt3-L polypeptides includeprokaryotes, yeast or higher eukaryotic cells. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts are described, for example, in Pouwels et al. CloningVectors: A Laboratory Manual, Elsevier, N.Y., (1985). Cell-freetranslation systems could also be employed to produce flt3-Lpolypeptides using RNAs derived from DNA constructs disclosed herein.

[0068] Prokaryotes include gram negative or gram positive organisms, forexample, E. coli or Bacilli. Suitable prokaryotic host cells fortransformation include, for example, E. coli, Bacillus subtilis,Salmonella typhimurium, and various other species within the generaPseudomonas, Streptomyces, and Staphylococcus. In a prokaryotic hostcell, such as E. coli, a flt3-L polypeptide may include an N-terminalmethionine residue to facilitate expression of the recombinantpolypeptide in the prokaryotic host cell. The N-terminal Met may becleaved from the expressed recombinant flt3-L polypeptide.

[0069] Expression vectors for use in prokaryotic host cells generallycomprise one or more phenotypic selectable marker genes. A phenotypicselectable marker gene is, for example, a gene encoding a protein thatconfers antibiotic resistance or that supplies an autotrophicrequirement. Examples of useful expression vectors for prokaryotic hostcells include those derived from commercially available plasmids such asthe cloning vector pBR322 (ATCC 37017). pBR322 contains genes forampicillin and tetracycline resistance and thus provides simple meansfor identifying transformed cells. To construct en expression vectorusing pBR322, an appropriate promoter and a flt3-L DNA sequence areinserted into the pBR322 vector. Other commercially available vectorsinclude, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala,Sweden) and pGEM1 (Promega Biotec, Madison, Wis., USA).

[0070] Promoter sequences commonly used for recombinant prokaryotic hostcell expression vectors include β-lactamase (penicillinase), lactosepromoter system (Chang et al., Nature 275:615, 1978; and Goeddel et al.,Nature 281:544, 1979), tryptophan (trp) promoter system (Goeddel et al.,Nucl. Acids Res. 8:4057, 1980; and EP-A-36776) and tac promoter(Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, p. 412, 1982). A particularly useful prokaryotic host cellexpression system employs a phage λ P_(L) promoter and a cI857tsthermolabile repressor sequence. Plasmid vectors available from theAmerican Type Culture Collection which incorporate derivatives of the λP_(L) promoter include plasmid pHUB2 (resident in E. coli strain JMB9(ATCC 37092)) and pPLc28 (resident in E. coli RR1 (ATCC 53082)).

[0071] Flt3-L polypeptides alternatively may be expressed in yeast hostcells, preferably from the Saccharomyces genus (e.g., S. cerevisiae).Other genera of yeast, such as Pichia , K. lactis or Kluyveromyces, mayalso be employed. Yeast vectors will often contain an origin ofreplication sequence from a 2μ yeast plasmid, an autonomouslyreplicating sequence (ARS), a promoter region, sequences forpolyadenylation, sequences for transcription termination, and aselectable marker gene. Suitable promoter sequences for yeast vectorsinclude, among others, promoters for metallothionein, 3-phosphoglyceratekinase (Hitzeman et al., J. Biol. Chem. 255:2073, 1980) or otherglycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7:149, 1968; andHolland et al., Biochem. 17:4900, 1978), such as enolase,glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. Other suitable vectors andpromoters for use in yeast expression are further described in Hitzeman,EPA-73,657 or in Fleer et. al., Gene, 107:285-195 (1991); and van denBerg et. al., Bio/Technology, 8:135-139 (1990). Another alternative isthe glucose-repressible ADH2 promoter described by Russell et al. (J.Biol. Chem. 258:2674, 1982) and Beier et al. (Nature 300:724, 1982).Shuttle vectors replicable in both yeast and E. coli may be constructedby inserting DNA sequences from pBR322 for selection and replication inE. coli (Amp^(r) gene and origin of replication) into theabove-described yeast vectors.

[0072] The yeast α-factor leader sequence may be employed to directsecretion of the flt3-L polypeptide. The α-factor leader sequence isoften inserted between the promoter sequence and the structural genesequence. See, e.g., Kurjan et al., Cell 30:933, 1982; Bitter et al.,Proc. Natl. Acad. Sci. USA 81:5330, 1984; U.S. Pat. No. 4,546,082; andEP 324,274. Other leader sequences suitable for facilitating secretionof recombinant polypeptides from yeast hosts are known to those of skillin the art. A leader sequence may be modified near its 3′ end to containone or more restriction sites. This will facilitate fusion of the leadersequence to the structural gene.

[0073] Yeast transformation protocols are known to those of skill in theart. One such protocol is described by Hinnen et al., Proc. Natl. Acad.Sci. USA 75:1929, 1978. The Hinnen et al. protocol selects for Trp⁺transformants in a selective medium, wherein the selective mediumconsists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose,10 μg/ml adenine and 20 μg/ml uracil.

[0074] Yeast host cells transformed by vectors containing ADH2 promotersequence may be grown for inducing expression in a “rich” medium. Anexample of a rich medium is one consisting of 1% yeast extract, 2%peptone, and 1% glucose supplemented with 80 μg/ml adenine and 80 μg/mluracil. Derepression of the ADH2 promoter occurs when glucose isexhausted from the medium.

[0075] Mammalian or insect host cell culture systems could also beemployed to express recombinant flt3-L polypeptides. Baculovirus systemsfor production of heterologous proteins in insect cells are reviewed byLuckow and Summers, Bio/Technology 6:47 (1988). Established cell linesof mammalian origin also may be employed. Examples of suitable mammalianhost cell lines include the COS-7 line of monkey kidney cells (ATCC CRL1651) (Gluzman et al., Cell 23:175, 1981), L cells, C127 cells, 3T3cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, andBHK (ATCC CRL 10) cell lines, and the CV-1/EBNA-1 cell line derived fromthe African green monkey kidney cell line CVI (ATCC CCL 70) as describedby McMahan et al. (EMBO J. 10:2821, 1991).

[0076] Transcriptional and translational control sequences for mammalianhost cell expression vectors may be excised from viral genomes. Commonlyused promoter sequences and enhancer sequences are derived from Polyomavirus, Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus.DNA sequences derived from the SV40 viral genome, for example, SV40origin, early and late promoter, enhancer, splice, and polyadenylationsites may be used to provide other genetic elements for expression of astructural gene sequence in a mammalian host cell. Viral early and latepromoters are particularly useful because both are easily obtained froma viral genome as a fragment which may also contain a viral origin ofreplication (Fiers et al., Nature 273:113, 1978). Smaller or larger SV40fragments may also be used, provided the approximately 250 bp sequenceextending from the Hind III site toward the Bgl I site located in theSV40 viral origin of replication site is included.

[0077] Exemplary expression vectors for use in mammalian host cells canbe constructed as disclosed by Okayama and Berg (Mol. Cell. Biol. 3:280,1983). A useful system for stable high level expression of mammaliancDNAs in C127 murine mammary epithelial cells can be constructedsubstantially as described by Cosman et al. (Mol. Immunol. 23:935,1986). A useful high expression vector, PMLSV N1/N4, described by Cosmanet al., Nature 312:768, 1984 has been deposited as ATCC 39890.Additional useful mammalian expression vectors are described inEP-A-0367566, and in U.S. Pat. application Ser. No. 07/701,415, filedMay 16, 1991, incorporated by reference herein. The vectors may bederived from retroviruses. In place of the native signal sequence, aheterologous signal sequence may be added, such as the signal sequencefor IL-7 described in U.S. Pat. No. 4,965,195; the signal sequence forIL-2 receptor described in Cosman et al., Nature 312:768 (1984); theIL-4 signal peptide described in EP 367,566; the type I IL-1 receptorsignal peptide described in U.S. Pat. No. 4,968,607; and the type IIIL-1 receptor signal peptide described in EP 460,846.

[0078] Flt3-L as an isolated or homogeneous protein according to theinvention may be produced by recombinant expression systems as describedabove or purified from naturally occurring cells. Flt3-L can be purifiedto substantial homogeneity, as indicated by a single protein band uponanalysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

[0079] One process for producing flt3-L comprises culturing a host celltransformed with an expression vector comprising a DNA sequence thatencodes flt3-L under conditions sufficient to promote expression offlt3-L. Flt3-L is then recovered from culture medium or cell extracts,depending upon the expression system employed. As is known to theskilled artisan, procedures for purifying a recombinant protein willvary according to such factors as the type of host cells employed andwhether or not the recombinant protein is secreted into the culturemedium.

[0080] For example, when expression systems that secrete the recombinantprotein are employed, the culture medium first may be concentrated usinga commercially available protein concentration filter, for example, anAmicon or Millipore Pellicon ultrafiltration unit. Following theconcentration step, the concentrate can be applied to a purificationmatrix such as a gel filtration medium. Alternatively, an anion exchangeresin can be employed, for example, a matrix or substrate having pendantdiethylaminoethyl (DEAE) groups. The matrices can be acrylamide,agarose, dextran, cellulose or other types commonly employed in proteinpurification. Alternatively, a cation exchange step can be employed.Suitable cation exchangers include various insoluble matrices comprisingsulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred.Finally, one or more reversed-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,(e.g., silica gel having pendant methyl or other aliphatic groups) canbe employed to further purify flt3-L. Some or all of the foregoingpurification steps, in various combinations, are well known and can beemployed to provide a substantially homogeneous recombinant protein.

[0081] It is possible to utilize an affinity column comprising theligand binding domain of flt3 receptors to affinity-purify expressedflt3-L polypeptides. Flt3-L polypeptides can be removed from an affinitycolumn using conventional techniques, e.g., in a high salt elutionbuffer and then dialyzed into a lower salt buffer for use or by changingpH or other components depending on the affinity matrix utilized.Alternatively, the affinity column may comprise an antibody that bindsflt3-L. Example 6 describes a procedure for employing flt3-L of theinvention to generate monoclonal antibodies directed against flt3-L.

[0082] Recombinant protein produced in bacterial culture is usuallyisolated by initial disruption of the host cells, centrifugation,extraction from cell pellets if an insoluble polypeptide, or from thesupernatant fluid if a soluble polypeptide, followed by one or moreconcentration, salting-out, ion exchange, affinity purification or sizeexclusion chromatography steps. Finally, RP-HPLC can be employed forfinal purification steps. Microbial cells can be disrupted by anyconvenient method, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents.

[0083] Transformed yeast host cells are preferably employed to expressflt3-L as a secreted polypeptide in order to simplify purification.Secreted recombinant polypeptide from a yeast host cell fermentation canbe purified by methods analogous to those disclosed by Urdal et al. (J.Chromatog. 296:171, 1984). Urdal et al. describe two sequential,reversed-phase HPLC steps for purification of recombinant human IL-2 ona preparative HPLC column.

[0084] Antisense or sense oligonucleotides comprising a single-strandednucleic acid sequence (either RNA or DNA) capable of binding to a targetflt3-L mRNA sequence (forming a duplex) or to the flt3-L sequence in thedouble-stranded DNA helix (forming a triple helix) can be made accordingto the invention. Antisense or sense oligonucleotides, according to thepresent invention, comprise a fragment of the coding region of flt3-LcDNA. Such a fragment generally comprises at least about 14 nucleotides,preferably from about 14 to about 30 nucleotides. The ability to createan antisense or a sense oligonucleotide, based upon a cDNA sequence fora given protein is described in, for example, Stein and Cohen, CancerRes. 48:2659, 1988 and van der Krol et al., BioTechniques 6:958, 1988.

[0085] Binding of antisense or sense oligonucleotides to target nucleicacid sequences results in the formation of complexes that blocktranslation (RNA) or transcription (DNA) by one of several means,including enhanced degradation of the duplexes, premature termination oftranscription or translation, or by other means. The antisenseoligonucleotides thus may be used to block expression of flt3-Lproteins. Antisense or sense oligonucleotides further compriseoligonucleotides having modified sugar-phosphodiester backbones (orother sugar linkages, such as those described in WO91/06629) and whereinsuch sugar linkages are resistant to endogenous nucleases. Sucholigonucleotides with resistant sugar linkages are stable in vivo (i.e.,capable of resisting enzymatic degradation) but retain sequencespecificity to be able to bind to target nucleotide sequences. Otherexamples of sense or antisense oligonucleotides include thoseoligonucleotides which are covalently linked to organic moieties, suchas those described in WO 90/10448, and other moieties that increasesaffinity of the oligonucleotide for a target nucleic acid sequence, suchas poly-(L-lysine). Further still, intercalating agents, such asellipticine, and alkylating agents or metal complexes may be attached tosense or antisense oligonucleotides to modify binding specificities ofthe antisense or sense oliginucleotide for the target nucleotidesequence.

[0086] Antisense or sense oligonucleotides may be introduced into a cellcontaining the target nucleic acid sequence by any gene transfer method,including, for example, CaPO₄-mediated DNA transfection,electroporation, or by using gene transfer vectors such as Epstein-Barrvirus. Antisense or sense oligonucleotides are preferably introducedinto a cell containing the target nucleic acid sequence by insertion ofthe antisense or sense oligonucleotide into a suitable retroviralvector, then contacting the cell with the retrovirus vector containingthe inserted sequence, either in vivo or ex vivo. Suitable retroviralvectors include, but are not limited to, the murine retrovirus M-MuLV,N2 (a retrovirus derived from M-MuLV), or or the double copy vectorsdesignated DCT5A, DCT5B and DCT5C (see PCT Application US 90/02656).

[0087] Sense or antisense oligonucleotides also may be introduced into acell containing the target nucleotide sequence by formation of aconjugate with a ligand binding molecule, as described in WO 91/04753.Suitable ligand binding molecules include, but are not limited to, cellsurface receptors, growth factors, other cytokines, or other ligandsthat bind to cell surface receptors. Preferably, conjugation of theligand binding molecule does not substantially interfere with theability of the ligand binding molecule to bind to its correspondingmolecule or receptor, or block entry of the sense or antisenseoligonucleotide or its conjugated version into the cell.

[0088] Alternatively, a sense or an antisense oligonucleotide may beintroduced into a cell containing the target nucleic acid sequence byformation of an oligonucleotide-lipid complex, as described in WO90/10448. The sense or antisense oligonucleotide-lipid complex ispreferably dissociated within the cell by an endogenous lipase.

[0089] Flt3-L polypeptides of the invention can be formulated accordingto known methods used to prepare pharmaceutically useful compositions.Flt3-L can be combined in admixture, either as the sole active materialor with other known active materials, with pharmaceutically suitablediluents (e.g., Tris-HCl, acetate, phosphate), preservatives (e.g.,Thimerosal, benzyl alcohol, parabens), emulsifiers, solubilizers,adjuvants and/or carriers. Suitable carriers and their formulations aredescribed in Remington's Pharmaceutical Sciences, 16th ed. 1980, MackPublishing Co. In addition, such compositions can contain flt3-Lcomplexed with polyethylene glycol (PEG), metal ions, or incorporatedinto polymeric compounds such as polyacetic acid, polyglycolic acid,hydrogels, etc., or incorporated into liposomes, microemulsions,micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts orspheroblasts. Such compositions will influence the physical state,solubility, stability, rate of in vivo release, and rate of in vivoclearance of flt3-L. Flt3-L can also be conjugated to antibodies againsttissue-specific receptors, ligands or antigens, or coupled to ligands oftissue-specific receptors. Where the flt3 receptor is found onneoplastic cells, the flt3-L may be conjugated to a toxin whereby flt3-Lis used to deliver the toxin to the specific site, or may be used tosensitize such neoplastic cells to subsequently administeredanti-neoplastic agents.

[0090] Flt3-L can be administered topically, parenterally, or byinhalation. The term “parenteral” includes subcutaneous injections,intravenous, intramuscular, intracisternal injection, or infusiontechniques. These compositions will typically contain an effectiveamount of the flt3-L, alone or in combination with an effective amountof any other active material. Such dosages and desired drugconcentrations contained in the compositions may vary depending uponmany factors, including the intended use, patient's body weight and age,and route of administration. Preliminary doses can be determinedaccording to animal tests, and the scaling of dosages for humanadministration can be performed according to art-accepted practices.Keeping the above description in mind, typical dosages of flt3-L mayrange from about 10 μg per square meter to about 1000 μg per squaremeter. A preferred dose range is on the order of about 100 μg per squaremeter to about 300 μg per square meter.

[0091] In addition to the above, the following examples are provided toillustrate particular embodiments and not to limit the scope of theinvention.

EXAMPLE 1 Preparation of Flt3-Receptor:Fc Fusion Protein

[0092] This example describes the cloning of murine flt3 cDNA, and theconstruction of an expression vector encoding a soluble murineflt3-receptor:Fc fusion protein for use in detecting cDNA clonesencoding flt3-L. Polymerase chain reaction (PCR) cloning of the flt3cDNA from a murine T-cell was accomplished using the oligonucleotideprimers and the methods as described by Lyman et al., Oncogene,8:815-822, (1993), incorporated herein by reference. The cDNA sequenceand encoded amino acid sequence for mouse flt3 receptor is presented byRosnet et el., Oncogene, 6:1641-1650, (1991), hereby incorporated byreference. The mouse flt3 protein has a 542 amino acid extracellulardomain, a 21 amino acid transmembrane domain, and a 437 amino acidcytoplasmic domain.

[0093] Prior to fusing the murine flt3 cDNA to the N-terminus of cDNAencoding the Fc portion of a human IgG1 molecule, the amplified mouseflt3 cDNA fragment was inserted into Asp718-NotI site of pCAV/NOT,described in PCT Application WO 90/05183. DNA encoding a single chainpolypeptide comprising the Fc region of a human IgG1 antibody was clonedinto the SpeI site of the pBLUESCRIPT SK® vector, which is commerciallyavailable from Stratagene Cloning Systems, La Jolla, Calif. This plasmidvector is replicable in E. coli and contains a polylinker segment thatincludes 21 unique restriction sites. A unique BglII site was introducednear the 5′ end of the inserted Fc encoding sequence, such that theBglII site encompasses the codons for amino acids three and four of theFc polypeptide.

[0094] The encoded Fc polypeptide extends from the N-terminal hingeregion to the native C-terminus, i.e., is an essentially full-lengthantibody Fc region. Fragments of Fc regions, e.g., those that aretruncated at the C-terminal end, also may be employed. The fragmentspreferably contain multiple cysteine residues (at least the cysteineresidues in the hinge reaction) to permit interchain disulfide bonds toform between the Fc polypeptide portions of two separate flt3:Fc fusionproteins, forming dimers as discussed above.

[0095] An Asp718 restriction endonuclease cleavage site was introducedupstream of the flt3 coding region. An Asp 718-NotI fragment of mouseflt3 cDNA (comprising the entire extracellular domain, the transmembraneregion, and a small portion of the cytoplasmic domain) was isolated. Theabove-described Asp718-NotI flt3 partial cDNA was cloned into thepBLUESCRIPT SK® vector containing the Fc cDNA, such that the flt3 cDNAis positioned upstream of the Fc cDNA. Single stranded DNA derived fromthe resulting gene fusion was mutagenized by the method described inKunkel (Proc. Natl. Acad. Sci. USA 82:488, 1985) and Kunkel et al.(Methods in Enzymol. 154:367, 1987) in order to perfectly fuse theentire extracellular domain of flt3 to the Fc sequence. The mutagenizedDNA was sequenced to confirm that the proper nucleotides had beenremoved (i.e., transmembrane region and partial cytoplasmic domain DNAwas deleted) and that the flt3 and Fc sequences were in the same readingframe. The fusion cDNA was then excised and inserted into a mammalianexpression vector designated sfHAV-EO 409 which was cut with SalI-NotI,and the SalI and Asp718 ends blunted. The sfHAV-EO vector (also known aspDC406) is described by McMahan et al. (EMBO J., 10; No. 10:2821-2832(1991)).

[0096] Flt3:Fc fusion proteins preferably are synthesized in recombinantmammalian cell culture. The flt3:Fc fusion-containing expression vectorwas transfected into CV-1 cells (ATCC CCL 70) and COS-7 cells (ATCC CRL1651), both derived from monkey kidney. Flt3:Fc expression level wasrelatively low in both CV-1 and COS-7 cells. Thus, expression in 293cells (transformed primary human embryonal kidney cells, ATCC CRL 1573)was attempted.

[0097] The 293 cells transfected with the sfHAV-EO/flt3:Fc vector werecultivated in roller bottles to allow transient expression of the fusionprotein, which is secreted into the culture medium via the flt3 signalpeptide. The fusion protein was purified on protein A Sepharose columns,eluted, and used to screen cells for the ability to bind flt3:Fc, asdescribed in Examples 2 and 3.

EXAMPLE 2 Screening Cells for Flt3:Fc Binding

[0098] Approximately 100 different primary cells and cell lines fallinginto the following general categories: primary murine fetal brain cells,murine fetal liver cell lines, rat fetal brain cell lines, human lungcarcinoma (fibroblastoid) cell lines, human and murine lymphoid andmyeloid cell lines were assayed for flt3:Fc binding. Cell lines wereincubated with flt3:Fc, followed by a biotinylated anti-human Fcantibody, followed by streptavidin-phycoerythrin (Becton Dickinson). Thebiotinylated antibody was purchased from Jackson ImmunoresearchLaboratories. Streptavidin binds to the biotin molecule attached to theanti-human Fc antibody, which in turn binds to the Fc portion of theflt3:Fc fusion protein. Phycoerythrin is a fluorescent phycobiliproteinwhich serves as a detectable label. The level of fluorescence signal wasmeasured for each cell type using a FACScan® flow cytometer (BectonDickinson). The cell types deemed positive for flt3 :Fc binding wereidentified.

EXAMPLE 3 Isolation and Cloning of Flt3 L cDNA from Murine T-Cell cDNALibrary

[0099] A murine T-cell cDNA library of cell line P7B-0.3A4 was chosen asa possible source of flt3-L cDNA. P7B-0.3A4 is a murine T cell clonethat is Thy1.2⁺, CD4⁻, CD8⁻, TCRab^(±), CD44⁺. It was originally clonedat a cell density of 0.33 cells/well in the presence of rHuIL-7 andimmobilized anti-CD3 MAb, and was grown in continuous culture for morethan 1 year by passage once a week in medium containing 15 ng/mlrHuIL-7. The parent cell line was derived from lymph node cells of SJL/Jmice immunized with 50 nmoles PLP₁₃₉₋₁₅₁ peptide and 100 μgMycobacterium tuberculosis H37Ra in Incomplete Freund's Adjuvant. PLP isthe proteolipid protein component of the myelin sheath of the centralnervous system. The peptide composed of amino acids 139-151 haspreviously been shown to be the encephalogenic peptide in experimentalautoimmune encephalomyelitis (EAE), a murine model for multiplesclerosis in SJL/J mice. (Touhy, V. K., Z. Lu, R. A. Sobel, R. A.Laursen and M. B. Lees; 1989. Identification of an encephalitogenicdeterminant of myelin proteolipid protein for SJL mice. J. Immunol.142:1523.) After the initial culture in the presence of antigen, theparent cell line, designated PLP7, had been in continuous culture withrHuIL-7 (and without antigen) for more than 6 months prior to cloning.

[0100] P7B-0.3A4 proliferates only in response to very highconcentrations of PLP₁₃₉₋₁₅₁ peptide in the presence of irradiatedsyngeneic splenocytes and is not encephalogenic or alloresponsive. Thisclone proliferates in response to immobilized anti-CD3 MAb, IL-2, andIL-7, but not IL-4.

[0101] Binding of flt3:Fc was observed on murine T-cells and humanT-cells, and therefore a murine T-cell line was chosen (0.3A4) due toits ease of growth. A murine 0.3A4 cDNA library in sfHAV-EO was preparedas described in McMahan et al. (EMBO J., 10; No:10; 2821-2832 1991).sfHAV-EO is a mammalian expression vector that also replicates in E.coli. sfHAV-EO contains origins of replication derived from SV40,Epstein-Barr virus and pBR322 and is a derivative of HAV-EO described byDower et al., J.Immunol. 142:4314 (1989). sfHAV-EO differs from HAV-EOby the deletion of the intron present in the adenovirus 2 tripartiteleader sequence in HAV-EO. Briefly, murine T-cell cDNA was cloned intothe SalI site of sfHAV-EO by an adaptor method similar to that describedby Haymerle et al (Nucl. Acids Res. 14:8615, 1986), using the followingoligonucleotide adapter pair: 5′ TCGACTGGAACGAGACGACCTGCT 3′ SEQ ID NO:33′     GACCTTGCTCTGCTGGACGA 5′ SEQ ID NO:4

[0102] Double-stranded, blunt-ended, random-primed cDNA was preparedfrom 0.3A4 poly (A)+ RNA essentially as described by Gubler and Hoffman,Gene, 25:263-269 (1983), using a Pharmacia DNA kit. The above adapterswere added to the cDNA as described by Haymerle et al.. Low molecularweight material was removed by passage over Sephacryl S-1000 at 65° C.,and the cDNA was ligated into sfHAV-EO410, which had previously been cutwith SalI and ligated to the same oligonucleotide pair. This vector isdesignated as sfHAV-EO410. DNA was electroporated (Dower et al., NucleicAcids Res., 16:6127-6145, (1988) into E. coli DH10B, and after one hourgrowth at 37° C., the transformed cells were frozen in one milliliteraliquots in SOC medium (Hanahan et al., J. Mol. Biol., 166:557-580,(1983) containing 20% glycerol. One aliquot was titered to determine thenumber of ampcillin-resistant colonies. The resulting 0.3A4 library had1.84 million clones.

[0103]E. coli strain DH10B cells transfected with the cDNA library insfHAV-EO410 were plated to provide approximately 1600 colonies perplate. Colonies were scraped from each plate, pooled, and plasmid DNAprepared from each pool. The pooled DNA, representing about 1600colonies, was then used to transfect a sub-confluent layer ofCV-1/EBNA-1 cells using DEAE-dextran followed by chloroquine treatment,similar to that described by Luthman et al., Nucl. Acids Res. 11:1295,(1983) and McCutchan et al., J. Natl. Cancer Inst. 41:351, (1986). TheCV-1/EBNA-1 cell line (ATCC CRL10478) constitutively expresses EBVnuclear antigen-1 driven from the CMV immediate-early enhancer/promoter.CV1-EBNA-1 was derived from the African Green Monkey kidney cell lineCV-1 (ATCC CCL 70), as described by McMahan et al. (EMBO J. 10:2821,1991).

[0104] In order to transfect the CV-1/EBNA-1 cells with the cDNAlibrary, the cells were maintained in complete medium (Dulbecco'smodified Eagle's media (DMEM) containing 10% (v/v) fetal calf serum(FCS), 50 U/ml penicillin, 50 U/ml streptomycin, 2 mM L-glutamine) andwere plated at a density of about 2×10⁵ cells/well on single-wellchambered slides (Lab-Tek). Slides were pretreated with 1 mil humanfibronectin (10 μg/ml in PBS) for 30 minutes followed by 1 wash withPBS. Media was removed from the adherent cell layer and replaced with1.5 ml complete medium containing 66.6 μM chloroquine sulfate.Two-tenths nil of DNA solution (2 μg DNA, 0.5 mg/ml DEAE-dextran incomplete medium containing chloroquine) was then added to the cells andincubated for 5 hours. Following the incubation, the media was removedand the cells shocked by addition of complete medium containing 10% DMSOfor 2.5 to 20 minutes followed by replacement of the solution with freshcomplete medium. The cells were cultured for 2 to 3 days to permittransient expression of the inserted sequences.

[0105] Transfected monolayers of CV-1/EBNA-1 cells were assayed forexpression of flt3-L by slide autoradiography essentially as describedby Gearing et al. (EMBO J. 8:3667, 1989). Transfected CV-1/EBNA-1 cells(adhered to chambered slides) were washed once with binding medium withnonfat dry milk (BM-NFDM) (RPMI medium 1640 containing 25 mg/ml bovineserum albumin (BSA), 2 mg/mil sodium azide, 20 mM HEPES, pH 7.2, and 50mg/ml nonfat dry milk). Cells were then incubated with flt3:Fc inBM-NFDM (1 μg/ml) for 1 hour at room temperature. After incubation, thecell monolayers in the chambered slides were washed three times withBM-NFDM to remove unbound flt3:Fc fusion protein and then incubated with40 ng/ml ¹²⁵I-mouse anti-human Fc antibody (described below) (a 1:50dilution) for 1 hour at room temperature. The cells were washed threetimes with BM-NFDM, followed by 2 washes with phosphate-buffered saline(PBS) to remove unbound ¹²⁵I-mouse anti-human Fc antibody. The cellswere fixed by incubating for 30 minutes at room temperature in 2.5%glutaraldehyde in PBS, pH 7.3, washed twice in PBS and air dried. Thechamber slides containing the cells were exposed on a Phophorimager(Molecular Dynamics) overnight, then dipped in Kodak GTNB-2 photographicemulsion (6× dilution in water) and exposed in the dark for 3-5 days at4° C. in a light proof box. The slides were then developed forapproximately 4 minutes in Kodak D19 developer (40 g/500 ml water),rinsed in water and fixed in Agfa G433C fixer. The slides wereindividually examined with a microscope at 25-40× magnification andpositive cells expressing flt3-L were identified by the presence ofautoradiographic silver grains against a light background.

[0106] The mouse anti-human Fc antibody was obtained from JacksonLaboratories. This antibody showed minimal binding to Fc proteins boundto the Fcγ receptor. The antibody was labeled using the Chloramine Tmethod. Briefly, a Sephadex G-25 column was prepared according to themanufacturer's instructions. The column was pretreated with 10 columnvolumes of PBS containing 1% bovine serum albumin to reduce nonspecificadsorption of antibody to the column and resin. Nonbound bovine serumalbumin was then washed from the column with 5 volumes of PBS lackingbovine serum albumin. In a microfuge tube 10 μg of antibody (dissolvedin 10 μl of PBS) was added to 50 μl of 50 mM sodium phosphate buffer (pH7.2) 2.0 mCi of carrier-free Na¹²⁵I was added and the solution was mixedwell. 15 μl of a freshly prepared solution of chloramine-T (2 mg/ml in0.1 M sodium phosphate buffer (pH 7.2)) was then added and the mixturewas incubated for 30 minutes at room temperature, and the mixture thenwas immediately applied to the column of Sephadex G-25. Theradiolabelled antibody was then eluted from the column by collecting100-150 μl fractions of eluate. Bovine serum albumin was added to theeluted fractions containing the radiolabeled antibody to a finalconcentration of 1%. Radioiodination yielded specific activities in therange of 5-10×10¹⁵ cpm/nmol protein.

[0107] Using the slide autoradiography approach, the approximately1,840,000 cDNAs were screened in pools of approximately 1,600 cDNAsuntil assay of one transfectant pool showed multiple cells clearlypositive for flt3:Fc binding. This pool was then partitioned into poolsof 500 and again screened by slide autoradiography and a positive poolwas identified. This pool was partitioned into pools of 100 and againscreened.

[0108] Individual colonies from this pool of 100 were screened until aclone (clone #6C) was identified which directed synthesis of a surfaceprotein with detectable flt3:Fc binding activity. This clone wasisolated, and its 0.88 kb cDNA insert was sequenced.

[0109] The nucleotide and encoded amino acid sequences of the codingregion of the murine flt3-ligand cDNA of clone #6C are presented in SEQID NOS: 1 and 2. The cDNA insert is 0.88 kb in length. The open-readingframe within this sequence could encode a protein of 231 amino acids.Thus, DNA and encoded amino acid sequences for the 231-amino acid openreading frame are presented in SEQ ID NOS: 1 and 2. The protein of SEQID NO: 2 is a type I transmembrane protein, with an N-terminal signalpeptide (amino acids 1 to 27), an extracellular domain (amino acids28-188) a transmembrane domain (amino acids 189-211) and a cytoplasmicdomain (amino acids 212-231). The predicted molecular weight of thenative protein following cleavage of the signal sequence is 23,164daltons. The mature protein has an estimated pI of 9.372. There are 56bp of 5′ noncoding sequence and 126 bp of 3′ non-coding sequenceflanking the coding region, including the added cDNA adapters. Theabove-described cloning procedure also produced a murine flt3 ligandclone #5H, which is identical to the #6C clone beginning at nucleotide49 and continuing through nucleotide 545 (corresponding to amino acid163) of SEQ ID NO: 1. The #5H clone completely differs from that pointonward, and represents an alternate splicing construct.

[0110] The vector sfHAV-EO410 containing the flt3-L cDNA in E. coliDH10B cells was deposited with the American Type Culture Collection,Rockville, Md., USA (ATCC) on Apr. 20, 1993 and assigned accessionnumber ATCC 69286. The deposit was made under the terms of the BudapestTreaty.

EXAMPLE 4 Cloning of cDNA Encoding Human Flt3-L

[0111] A cDNA encoding human flt3-L was cloned from a human clone 22 Tcell λgt10 random primed cDNA library as described by Sims et al., PNAS,86:8946-8950 (1989). The library was screened with a 413 bp Ple Ifragment corresponding to the extracellular domain of the murine flt3-L(nucleotides 103-516 of SEQ ID NO: 1). The fragment was random primed,hybridized overnight to the library filters at 55° C. in oligoprehybridization buffer. The fragment was then washed at 55° C. at2×SSC/0.1% SDS for one hour, followed by 1×SSC/0.1% SDS for one hour andthen by 0.5×SSC/0,1% SDS for one hour. The DNA from the positive phageplaques was extracted, and the inserts were amplified by PCR usingoligonucleotides specific for the phage arms. The DNA then wassequenced, and the sequence for clone #9 is shown in SEQ ID NO: 5.Additional human flt3-L cDNA was isolated from the same λgt10 randomprimed cDNA library as described above by screening the library with afragment of the extracellular domain of the murine clone #5H cDNAcomprising a cDNA sequence essentially corresponding to nucleotides128-541 of SEQ ID NO: 1.

[0112] Sequencing of the 988 bp cDNA clone #9 revealed an open readingframe of 705 bp surrounded by 29 bp of 5′ non-coding sequence and 250 bpof 3′ non-coding sequence. The 3′ non-coding region did not contain apoly-A tail. There were no in-frame stop codons upstream of theinitiator methionine. The open reading frame encodes a type Itransmembrane protein of 235 amino acids as shown by amino acids 1-235of SEQ ID NO: 6. The protein has an N-terminal signal peptide ofalternatively 26 or 27 amino acids. There exists a slightly greaterprobability that the N-terminal signal peptide is 26 amino acids inlength than 27 amino acids in length. The signal peptide is followed bya 156 or a 155 amino acid extracellular domain (for signal peptides of26 and 27 amino acids, respectively); a 23 amino acid transmembranedomain and a 30 amino acid cytoplasmic domain. Human flt3-L sharesoverall 72% amino acid identity and 78% amino acid similarity withmurine flt3-L. The vector pBLUESCRIPT SK(−) containing the human flt3-LcDNA of clone #9 was deposited with the American Type CultureCollection, Rockville, Md., USA (ATCC) on Aug. 6, 1993 and assignedacession number ATCC 69382. The deposit was made under the terms of theBudapest Treaty.

EXAMPLE 5 Expression of Flt3-L in Yeast

[0113] For expression of soluble flt3-L in yeast, syntheticoligonucleotide primers were used to amplify via PCR (Mullis andFaloona, Meth. Enzymol. 155:335-350, 1987) the entire extracellularcoding domain of flt3-L between the end of the signal peptide and thestart of the transmembrane segment. The 5′ primer(5-AATTGGTACCTTTGGATAAAAGAGACTACAAGGACGACGATGACAAGACACCTGACTGTTACTTCAGCCAC-3′) SEQ ID NO: 7 encoded a portion of of the alpha factor leader andan antigenic octapeptide, the FLAG sequence fused in-frame with thepredicted mature N-terminus of flt3-L. The 3′ oligonucleotide(5′-ATATGGATCCCTACTGCCTGGGCCGAGGCTCTGGGAG-3′) SEQ ID NO: 8 created atermination codon following Gin-189, just at the putative transmembraneregion. The PCR-generated DNA fragment was ligated into a yeastexpression vector (for expression in K. lactis) that directs secretionof the recombinant product into the yeast medium (Fleer et. al., Gene,107:285-195 (1991); and van den Berg et. al., Bio/Technology, 8:135-139(1990)). The FLAG:flt3-L fusion protein was purified from yeast broth byaffinity chromotography as previously described (Hopp et. al.,Biotechnology, 6:1204-1210, 1988).

EXAMPLE 6 Monoclonal Antibodies to Flt3-L

[0114] This example illustrates a method for preparing monoclonalantibodies to flt3-L. Flt3-L is expressed in mammalian host cells suchas COS-7 or CV- 1/EBNA-1 cells and purified using flt3:Fc affinitychromatography. Purified flt3-L, a fragment thereof such as theextracellular domain, synthetic peptides or cells that express flt3-Lcan be used to generate monoclonal antibodies against flt3-L usingconventional techniques, for example, those techniques described in U.S.Pat. No. 4,411,993. Briefly, mice are immunized with flt3-L as animmunogen emulsified in complete Freund's adjuvant, and injected inamounts ranging from 10-100 μg subcutaneously or intraperitoneally. Tento twelve days later, the immunized animals are boosted with additionalflt3-L emulsified in incomplete Freund's adjuvant. Mice are periodicallyboosted thereafter on a weekly to bi-weekly immunization schedule. Serumsamples are periodically taken by retro-orbital bleeding or tail-tipexcision to test for flt3-L antibodies by dot blot assay, ELISA(Enzyme-Linked Immunosorbent Assay) or inhibition of flt3 binding.

[0115] Following detection of an appropriate antibody titer, positiveanimals are provided one last intravenous injection of flt3-L in saline.Three to four days later, the animals are sacrificed, spleen cellsharvested, and spleen cells are fused to a murine myeloma cell line,e.g., NS1 or preferably P3×63Ag8.653 (ATCC CRL 1580). Fusions generatehybridoma cells, which are plated in multiple microtiter plates in a HAT(hypoxanthine, aminopterin and thymidine) selective medium to inhibitproliferation of non-fused cells, myeloma hybrids, and spleen cellhybrids.

[0116] The hybridoma cells are screened by ELISA for reactivity againstpurified flt3-L by adaptations of the techniques disclosed in Engvall etal., Immunochem. 8:871, 1971 and in U.S. Pat. No. 4,703,004. A preferredscreening technique is the antibody capture technique described inBeckmann et al., (J. Immunol. 144:4212, 1990) Positive hybridoma cellscan be injected intraperitoneally into syngeneic BALB/c mice to produceascites containing high concentrations of anti-flt3-L-L monoclonalantibodies. Alternatively, hybridoma cells can be grown in vitro inflasks or roller bottles by various techniques. Monoclonal antibodiesproduced in mouse ascites can be purified by ammonium sulfateprecipitation, followed by gel exclusion chromatography. Alternatively,affinity chromatography based upon binding of antibody to protein A orprotein G can also be used, as can affinity chromatography based uponbinding to flt3-L.

EXAMPLE 7 Use of Flt3-L Alone and in Combination with IL-7 or IL-3

[0117] This example demonstrates the stimulation and proliferation ofAA4.1⁺ fetal liver cells by compositions containing flt3-L and IL-7; aswell as the stimulation and proliferation of c-kit-positive (c-kit⁺)cells by compositions containing flt3-L and IL-3.

[0118] AA4.1-positive (AA4.1⁺) expressing cells were isolated from thelivers of day 14 fetal C57BL/6 mice by cell panning in Optilux 100 mmplastic Petri dishes (Falcon No. 1001, Oxnard, Calif.). Plates werecoated overnight at 4° C. in PBS plus 0.1% fetal bovine serum (FBS)containing 10 μg/ml AA4.1 antibody (McKearn et. al., J. Immunol.,132:332-339, 1984) and then washed extensively with PBS plus 1% FBSprior to use. A single cell suspension of liver cells was added at 10⁷cells/dish in PBS plus 1% FBS and allowed to adhere to the plates fortwo hours at 4° C. The plates were then extensively washed, and theadhering cells were harvested by scraping for analysis or further use inthe hematopoiesis assays described below. FACS analysis using AA4.1antibody demonstrated a >95% AA4.1⁺ cell population.

[0119] C-kit⁺ pluripotent stem cells were purified from adult mouse bonemarrow (de Vries et. al., J. Exp. Med., 176:1503-1509, 1992; and Visserand de Vries, Methods in Cell Biol., 1993, submitted). Low density cells(≦1.078 g/cm³) positive for the lectin wheat germ agglutinin andnegative for the antigens recognized by the B220 and 15-1.4.1 (Visseret. al., Meth. in Cell Biol., 33:451-468, 1990) monoclonal antibodies,could be divided into sub-populations of cells that do and do notexpress c-kit by using biotinylated Steel factor. The c-kit⁺ fractionhas been shown to contain pluripotent hematopoietic stem cells (de Vrieset. al., Science 255:989-991, 1992; Visser and de Vries, Methods in CellBiol., 1993, submitted; and Ware et. al., 1993, submitted).

[0120] AA4.1+ Fetal liver cells were cultured in recombinant IL-7 (U.S.Pat. No. 4,965,195) at 100 ng/ml and recombinant flt3-L at 250 ng/ml.Flt3-L was used in three different forms in the experiments: (1) aspresent on fixed, flt3-L-transfected CV1/EBNA cells; (2) as concentratedculture supernatants from these same flt3-L-transfected CV1/EBNA cells;and (3) as a purified and isolated polypeptide preparation from yeastsupernatant as described in Example 5.

[0121] Hematopoiesis Assays

[0122] The proliferation of c-kit⁺ stem cells, fetal liver AA4.1⁺ cellswas assayed in [3H]-thymidine incorporation assays as essentiallydescribed by deVries et. al., J. Exp. Med., 173:1205-1211, 1991.Purified c-kit⁺ stem cells were cultured at 37° C. in a fully humidifiedatmosphere of 6.5% CO₂ and 7% O₂ in air for 96 hours. Murine recombinantIL-3 was used at a final concentration of 100 ng/ml. Subsequently, thecells were pulsed with 2 μCi per well of [³H]-thymidine (81 Ci/mmol;Amersham Corp., Arlington Heights, Ill.) and incubated for an additional24 hours. AA4.1⁺ cells (approximately 20,000 cells/well) were incubatedin IL-7, flt3-L and flt3-L +IL-7 for 48 hours, followed by[³H]-thymidine pulse of six hours. The results of flt3-L and IL-7 areshown in Table I, and results of flt3-L and IL-3 are shown in Table II.TABLE 1 Effect of Flt3-L and IL-7 on Proliferation of AA4.1 + FetalLiver Cells. Factor Control flt3-L IL-7 flt3-L + IL-7 [³H]-thymidine 1001000 100 4200 incorporation (CPM)

[0123] The combination of flt3-L and IL-7 produced a response that wasapproximately four-fold greater than flt3-L alone and approximately40-fold greater than IL-7 alone. TABLE II Effect of Flt3-L and IL-3 onProliferation of C-kit + Cells. Factor Control (vector alone) flt3-LIL-3 flt3-L + IL-3 [³H]-thymidine 100 1800 3000 9100 incorporation (CPM)

[0124] Culture supernatant from CV1/EBNA cells transfected with flt3-LcDNA stimulated the proliferation of c-kit⁺ stem cells approximately18-fold greater than the culture supernatant of CV1/EBNA cellstransfected with the expression vector alone. Addition of IL-3 to flt3-Lcontaining supernatant showed a synergistic effect, with approximatelytwice the degree of proliferation observed than would be expected if theeffects were additive.

EXAMPLE 8 Construction of Flt3-L:Fc Fusion Protein

[0125] This example describes a methof for constructing a fusion proteincomprising an extracellular region of the flt3-L and the Fc domain of ahuman immunoglobulin. The methods are essentially the same as thosedescribed in Example 1 for construction of a flt3:Fc fusion protein.

[0126] Prior to fusing a flt3-L cDNA to the N-terminus of cDNA encodingthe Fc portion of a human IgG1 molecule, the flt3-L cDNA fragment isinserted into Asp718-NotI site of pCAV/NOT, described in PCT ApplicationWO 90/05183. DNA encoding a single chain polypeptide comprising the Fcregion of a human IgG1 antibody is cloned into the SpeI site of thepBLUESCRIPT SK® vector, which is commercially available from StratageneCloning Systems, La Jolla, Calif. This plasmid vector is replicable inE. coli and contains a polylinker segment that includes 21 uniquerestriction sites. A unique BglII site is then introduced near the 5′end of the inserted Fc encoding sequence, such that the BglII siteencompasses the codons for amino acids three and four of the Fcpolypeptide.

[0127] The encoded Fc polypeptide extends from the N-terminal hingeregion to the native C-terminus, i.e., is an essentially full-lengthantibody Fc region. Fragments of Fc regions, e.g., those that aretruncated at the C-terminal end, also may be employed. The fragmentspreferably contain multiple cysteine residues (at least the cysteineresidues in the hinge reaction) to permit interchain disulfide bonds toform between the Fc polypeptide portions of two separate flt3-L:Fcfusion proteins, forming dimers.

[0128] An Asp718-StuI partial cDNA of flt3-L in pCAV/NOT can be clonedinto a Asp718-SpeI site of pBLUESCRIPT SK® vector containing the FccDNA, such that the flt3-L cDNA is positioned upstream of the Fc cDNA.The sequence of single stranded DNA derived from the resulting genefusion can be affected by template-directed mutagensis described byKunkel (Proc. Natl. Acad. Sci. USA 82:488, 1985) and Kunkel et al.(Methods in Enzymol. 154:367, 1987) in order to perfectly fuse theentire extracellular domain of flt3-L to the Fc sequence. The resultingDNA can then be sequenced to confirm that the proper nucleotides areremoved (i.e., transmembrane region and partial cytoplasmic domain DNAare deleted) and that flt3-L and Fc sequences are in the same readingframe. The fusion cDNA is then excised and inserted using conventionalmethods into the mammalian expression vector pCAV/NOT which is cut withAsp 718-NotI.

[0129] Flt3-L:Fc fusion proteins preferably are synthesized inrecombinant mammalian cell culture. The flt3-L:Fc fusion-containingexpression vector is then transfected into CV-1 cells (ATCC CCL 70) orCOS-7 cells (ATCC CRL 1651). Expression in 293 cells (transformedprimary human embryonal kidney cells, ATCC CRL 1573) also is feasible.

[0130] The 293 cells transfected with the pCAV/NOT/flt3-L:Fc vector arecultivated in roller bottles to allow transient expression of the fusionprotein, which is secreted into the culture medium via the flt3-L signalpeptide. The fusion protein can be purified on protein A Sepharosecolumns.

EXAMPLE 9 Generation of Transgenic Mice That Overexpress Flt3-L

[0131] This example describes a procedure used to generate transgenicmice that overexpress flt3-L. Flt3-L-overexpressing transgenic mice werestudied to determine the biological effects of overexpression. Mouse(B16/J) pronuclei were microinjected with flt3-L DNA according to themethod described by Gordon et al., Science 214:1244-1246, (1981). Ingeneral, fertilized mouse eggs having visible pronuclei were firstplaced on an injection chamber and held in place with a small pipet. Aninjection pipet was then used to inject the gene encoding the flt3-L(clone #6C) into the pronuclei of the egg. Injected eggs were theneither (i) transferred into the oviduct of a 0.5 day p.c. pseudopregnantfemale; (ii) cultured in vitro to the two-cell stage (overnight) andtransferred into the oviduct of a 0.5 day p.c. pseudopregnant female; or(iii) cultured in vitro to the blastocyst stage and transferred into theuterus of a 2.5 day p.c. pseudopregnant female. Preferably, either ofthe first two options can be used since they avoid extended in vitroculture, and preferably, approximately 20-30 microinjected eggs shouldbe transferred to avoid small litters.

EXAMPLE 10 Flt3-L Stimulates Proliferation of Erythroid Cells in theSpleen

[0132] This example describes the effect of flt3-L on the production oferythroid cells in the spleen of transgenic mice. Transgenic mice weregenerated according to the procedures of Example 10. The mice weresacrificed and each intact spleen was made into a single cellsuspension. The suspended cells were spun and then resuspended in 10 nilof medium that contained PBS+1% fetal bovine serum. Cell counts wereperformed thereon using a hemocytometer. Each cell specimen was countedwith Trypan Blue stain to obtain a total viable cell count permilliliter of medium according to the following formula: (RBC+WBC)/ml,wherein RBC is the red blood cell count and WBC means the white bloodcell count. Each specimen then was counted with Turk's stain to obtain atotal white blood cell count per milliliter of medium. The total redblood cell count per milliliter was calculated for each specimen bysubtracting the total white blood cell count per milliliter from thetotal viable cell count per milliliter. The results are shown in thefollowing Table III. TABLE III Erythroid Cell Proliferation inFlt3-L-Overexpressing Transgenic Mice Spleen Total Viable Cell (millioncells/ Total White Cell Total Red Blood Cell Mouse ml) (millioncells/ml) (million cells/ml) Control 1 29.7 27 2.7 Control 2 31 24.6 6.4Transgenic 1 44.7 25.6 19.1 Transgenic 2 37.3 28.4 8.9

[0133] From the data of Table III, the white blood cell counts permilliliter were approximately the same as the control mice. However, thered blood cell counts from the spleens of the two transgenic mice wereapproximately two to three-fold greater than observed in the controlmice. Flt3-L stimulates an increase in cells of the erythroid lineage,possibly through stimulation of erythroid proogenitor cells, through thestimulation of cells that produce erythropoietin, or by blocking amechanism that inhibits erythropoiesis.

EXAMPLE 11 Flt3-L Stimulates Proliferation of T Cells and Early B Cells

[0134] Bone marrow from 9 week old transgenic mice generated accordingto Example 10 was screened for the presence of various T and B cellphenotype markers using antibodies that are immunoreactive with suchmarkers. The following markers were investigated: the B220 marker, whichis specific to the B cell lineage; surface IgM marker (sIgM), which isspecific to mature B cells; the S7 (CD43) marker, which is an early Bcell marker; the Stem Cell Antigen-1 (SCA-1) marker, which is a markerof activated T cells and B cells; CD4, which is a marker for helper Tcells and some stem cells; and the Mac-1 marker, which is specific tomacrophages, were screened using well known antibodies against suchmarkers. The following Table IV shows the data obtained from screeningthe bone marrow. Two transgenic mice from the same litter were analyzedagainst a normal mouse from the same litter (control), and an unrelatednormal mouse (control). and then to stage 3, characterized by cellshaving Pgp-1^(±), HSA⁺⁺, and IL-2R-markers. Thymic cells in stage 2 ofthe transgenic mice were reduced by about 50%, while the population ofcells in stage 3 was proportionately increased. These data suggest thatflt3-L drives the thymic cells from stage 2 to stage 3 of development,indicating that flt3-L is active on early T cells.

EXAMPLE 13 Use of Flt3-L in Peripheral Stem Cell Transplantation

[0135] This Example describes a method for using flt3-L in autologousperipheral stem cell (PSC) or peripheral blood progenitor cell (PBPC)transplantation. Typically, PBPC and PSC transplantation is performed onpatients whose bone marrow is unsuitable for collection due to, forexample, marrow abnormality or malignant involvement.

[0136] Prior to cell collection, it may be desirable to mobilize orincrease the numbers of circulating PBPC and PSC. Mobilization canimprove PBPC and PSC collection, and is achievable through theintravenous administration of flt3-L to the patients prior to collectionof such cells. Other growth factors such as CSF-1, GM-CSF, SF, G-CSF,EPO, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,IL-12, IL-13, IL-14, IL-15, GM-CSF/IL-3 fusion proteins, LIF, FGF andcombinations thereof, can be likewise administered in sequence, or inconcurrent combination with flt3-L. Mobilized or non-mobilized PBPC andPSC are collected using apheresis procedures known in the art. See, forexample, Bishop et al., Blood, vol. 83, No. 2, pp. 610-616 (1994).Briefly, PBPC and PSC are collected using conventional devices, forexample, a Haemonetics Model V50 apheresis device (Haemonetics,Braintree, Mass.). Four-hour collections are performed typically no morethan five times weekly until approximately 6.5×10⁸ mononuclear cells(MNC)/kg patient are collected. Aliquots of collected PBPC and PSC areassayed for granulocyte-macrophage colony-forming unit (CFU-GM) contentby diluting approximately 1:6 with Hank's balanced salt solution withoutcalcium or magnesium (HBSS) and layering over lymphocyte separationmedium (Organon Teknika, Durham, N.C.). Following centrifugation, MNC atthe interface are collected, washed and resuspended in HBSS. Onemilliliter aliquots containing approximately 300,000 MNC, modifiedMcCoy's 5A medium, 0.3% agar, 200 U/mL recombinant human GM-CSF, 200u/mL recombinant human IL-3, and 200 u/mL recombinant human G-CSF arecultured at 37° C. in 5% CO₂ in fully humidified air for 14 days.Optionally, flt3-L or GM-CSF/IL-3 fusion molecules (PIXY 321) may TABLEIV Effect of flt3-L Overexpression in Transgenic Mice Percentage ofPositive Cells Unrelated Littermate Marker Control Control Transgenic #1Transgenic #2 B220 30.64 27.17 45.84 48.78 sIgM 3.54 2.41 1.94 1.14S7(CD43) 54.43 45.44 46.11 50.59 SCA-1 10.92 11.74 19.45 27.37 CD4 6.948.72 12.21 14.05 Mac-1 36.80 27.15 21.39 18.63

[0137] The above data indicate that flt3-L overexpression in mice leadsto an increase in the number of B cells, as indicated by the increaseB220⁺ cells and SCA-1⁺ cells. Analysis of B220⁺ cells by FACS indicatedan increase in proB cells (HSA⁻, S7⁺). The increase in CD4⁺ cellsindicated an approximate two-fold increase in T cells and stem cells.The decrease in cells having the sIgM marker indicated that flt3-L doesnot stimulate proliferation of mature B cells. These data indicate thatflt3-L increases cells with a stem cell, T cell or an early B cellphenotype, and does not stimulate proliferation of mature B cells ormacrophages.

EXAMPLE 12 Analysis of the Thymus From Flt3-L-Over-expressing Mice

[0138] This Example describes the analysis of the thymus from thetransgenic mice generated according to the procedure of Example 10. Sixadult mice, each approximately three months of age, were sacrificed. Thethymus from each mouse was removed and a single cell suspension wasmade.

[0139] FACS analysis demonstrated that no total change in cell numberoccurred and that the mice showed no change in the ratios of maturingthymocytes using the markers: CD4 vs. CD8; CD3 vs. αβTCR (T cellreceptor); and CD3 vs. γδTCR (T cell receptor). However, a change in theratios of certain cell types within the CD4⁻ and CD8⁻ compartment (i.e.,the earliest cells with respect to development; which representapproximately 2% to 3% of total thymus cells) occurred. Specifically,CD4⁻ and CD8⁻ cells in the thymus develop in three stages. Stage 1represents cells having the Pgp-1⁺⁺, HSA⁺ and IL-2 receptor-negative(“IL-2R⁻”) markers. After stage 1, thymic cells develop to stage 2consisting of cells having Pgp-1⁺, HSA⁺⁺, and IL-2R⁺⁺ markers, be addedto the cultures. These cultures are stained with Wright's stain, andCFU-GM colonies are scored using a dissecting microscope (Ward et al.,Exp. Hematol., 16:358 (1988). Alternatively, CFU-GM colonies can beassayed using the CD34/CD33 flow cytometry method of Siena et al.,Blood, Vol. 77, No. 2, pp 400-409 (1991), or any other method known inthe art.

[0140] CFU-GM containing cultures are frozen in a controlled ratefreezer (e.g., Cryo-Med, Mt. Clemens, Mich.), then stored in the vaporphase of liquid nitrogen. Ten percent dimethylsulfoxide can be used as acryoprotectant. After all collections from the patient have been made,CFU-GM containing cultures are thawed and pooled. The thawed cellcollection either is reinfused intravenoulsy to the patient or expandedex vivo prior to reinfusion. Ex vivo expansion of pooled cells can beperformed using flt3-L as a growth factor either alone, sequentially orin concurrent combination with other cytokines listed above. Methods ofsuch ex vivo expansion are well known in the art. The cells, eitherexpanded or unexpanded, are reinfused intravenously to the patient. Tofacilitate engraftment of the transplanted cells, flt3-L is administeredsimultaneously with, or subsequent to, the reinfusion. Suchadministration of flt3-L is made alone, sequentially or in concurrentcombination with other cytokines selected from the list above.

EXAMPLE 14 Purification of Hematopoietic Progenitor and Stem Cells UsingFlt3-L

[0141] This Example describes a method for purifying hematopoieticprogenitor cells and stem cells from a suspension containing a mixtureof cells. Cells from bone marrow and peripheral blood are collectedusing conventional procedures. The cells are suspended in standard mediaand then centrifuged to remove red blood cells and neutrophils. Cellslocated at the interface between the two phases (also known in the artas the buffy coat) are withdrawn and resuspended. These cells arepredominantly mononuclear and represent a substantial portion of theearly hematopoietic progenitor and stem cells. The resulting cellsuspension then is incubated with biotinylated flt3-L for a sufficienttime to allow substantial flt3:flt3-L interaction. Typically, incubationtimes of at least one hour are sufficient. After incubation, the cellsuspension is passed, under the force of gravity, through a columnpacked with avidin-coated beads. Such columns are well known in the art,see Berenson, et al., J. Cell Biochem., 10D:239 (1986). The column iswashed with a PBS solution to remove unbound material. Target cells canbe released from the beads and from flt3-L using conventional methods.

1 8 879 base pairs nucleic acid single linear cDNA to mRNA NO NOmisc_feature 1..25 misc_feature 855..879 CDS 57..752 1 GTCGACTGGAACGAGACGAC CTGCTCTGTC ACAGGCATGA GGGGTCCCCG GCAGAG 56 ATG ACA GTG CTGGCG CCA GCC TGG AGC CCA AAT TCC TCC CTG TTG CTG 104 Met Thr Val Leu AlaPro Ala Trp Ser Pro Asn Ser Ser Leu Leu Leu 1 5 10 15 CTG TTG CTG CTGCTG AGT CCT TGC CTG CGG GGG ACA CCT GAC TGT TAC 152 Leu Leu Leu Leu LeuSer Pro Cys Leu Arg Gly Thr Pro Asp Cys Tyr 20 25 30 TTC AGC CAC AGT CCCATC TCC TCC AAC TTC AAA GTG AAG TTT AGA GAG 200 Phe Ser His Ser Pro IleSer Ser Asn Phe Lys Val Lys Phe Arg Glu 35 40 45 TTG ACT GAC CAC CTG CTTAAA GAT TAC CCA GTC ACT GTG GCC GTC AAT 248 Leu Thr Asp His Leu Leu LysAsp Tyr Pro Val Thr Val Ala Val Asn 50 55 60 CTT CAG GAC GAG AAG CAC TGCAAG GCC TTG TGG AGC CTC TTC CTA GCC 296 Leu Gln Asp Glu Lys His Cys LysAla Leu Trp Ser Leu Phe Leu Ala 65 70 75 80 CAG CGC TGG ATA GAG CAA CTGAAG ACT GTG GCA GGG TCT AAG ATG CAA 344 Gln Arg Trp Ile Glu Gln Leu LysThr Val Ala Gly Ser Lys Met Gln 85 90 95 ACG CTT CTG GAG GAC GTC AAC ACCGAG ATA CAT TTT GTC ACC TCA TGT 392 Thr Leu Leu Glu Asp Val Asn Thr GluIle His Phe Val Thr Ser Cys 100 105 110 ACC TTC CAG CCC CTA CCA GAA TGTCTG CGA TTC GTC CAG ACC AAC ATC 440 Thr Phe Gln Pro Leu Pro Glu Cys LeuArg Phe Val Gln Thr Asn Ile 115 120 125 TCC CAC CTC CTG AAG GAC ACC TGCACA CAG CTG CTT GCT CTG AAG CCC 488 Ser His Leu Leu Lys Asp Thr Cys ThrGln Leu Leu Ala Leu Lys Pro 130 135 140 TGT ATC GGG AAG GCC TGC CAG AATTTC TCT CGG TGC CTG GAG GTG CAG 536 Cys Ile Gly Lys Ala Cys Gln Asn PheSer Arg Cys Leu Glu Val Gln 145 150 155 160 TGC CAG CCG GAC TCC TCC ACCCTG CTG CCC CCA AGG AGT CCC ATA GCC 584 Cys Gln Pro Asp Ser Ser Thr LeuLeu Pro Pro Arg Ser Pro Ile Ala 165 170 175 CTA GAA GCC ACG GAG CTC CCAGAG CCT CGG CCC AGG CAG CTG TTG CTC 632 Leu Glu Ala Thr Glu Leu Pro GluPro Arg Pro Arg Gln Leu Leu Leu 180 185 190 CTG CTG CTG CTG CTG CCT CTCACA CTG GTG CTG CTG GCA GCC GCC TGG 680 Leu Leu Leu Leu Leu Pro Leu ThrLeu Val Leu Leu Ala Ala Ala Trp 195 200 205 GGC CTT CGC TGG CAA AGG GCAAGA AGG AGG GGG GAG CTC CAC CCT GGG 728 Gly Leu Arg Trp Gln Arg Ala ArgArg Arg Gly Glu Leu His Pro Gly 210 215 220 GTG CCC CTC CCC TCC CAT CCCTAGGATTCGA GCCTTGTGCA TCGTTGACTC 779 Val Pro Leu Pro Ser His Pro 225 230AGCCAGGGTC TTATCTCGGT TACACCTGTA ATCTCAGCCC TTGGGAGCCC AGAGCAGGAT 839TGCTGAATGG TCTGGAGCAG GTCGTCTCGT TCCAGTCGAC 879 231 amino acids aminoacid linear protein 2 Met Thr Val Leu Ala Pro Ala Trp Ser Pro Asn SerSer Leu Leu Leu 1 5 10 15 Leu Leu Leu Leu Leu Ser Pro Cys Leu Arg GlyThr Pro Asp Cys Tyr 20 25 30 Phe Ser His Ser Pro Ile Ser Ser Asn Phe LysVal Lys Phe Arg Glu 35 40 45 Leu Thr Asp His Leu Leu Lys Asp Tyr Pro ValThr Val Ala Val Asn 50 55 60 Leu Gln Asp Glu Lys His Cys Lys Ala Leu TrpSer Leu Phe Leu Ala 65 70 75 80 Gln Arg Trp Ile Glu Gln Leu Lys Thr ValAla Gly Ser Lys Met Gln 85 90 95 Thr Leu Leu Glu Asp Val Asn Thr Glu IleHis Phe Val Thr Ser Cys 100 105 110 Thr Phe Gln Pro Leu Pro Glu Cys LeuArg Phe Val Gln Thr Asn Ile 115 120 125 Ser His Leu Leu Lys Asp Thr CysThr Gln Leu Leu Ala Leu Lys Pro 130 135 140 Cys Ile Gly Lys Ala Cys GlnAsn Phe Ser Arg Cys Leu Glu Val Gln 145 150 155 160 Cys Gln Pro Asp SerSer Thr Leu Leu Pro Pro Arg Ser Pro Ile Ala 165 170 175 Leu Glu Ala ThrGlu Leu Pro Glu Pro Arg Pro Arg Gln Leu Leu Leu 180 185 190 Leu Leu LeuLeu Leu Pro Leu Thr Leu Val Leu Leu Ala Ala Ala Trp 195 200 205 Gly LeuArg Trp Gln Arg Ala Arg Arg Arg Gly Glu Leu His Pro Gly 210 215 220 ValPro Leu Pro Ser His Pro 225 230 24 base pairs nucleic acid single linearNO NO 3 TCGACTGGAA CGAGACGACC TGCT 24 20 base pairs nucleic acid singlelinear NO NO 4 AGCAGGTCGT CTCGTTCCAG 20 988 base pairs nucleic acidsingle linear cDNA to mRNA NO NO CDS 30..734 5 CGGCCGGAAT TCCGGGGCCCCCGGCCGAA ATG ACA GTG CTG GCG CCA GCC TGG 53 Met Thr Val Leu Ala Pro AlaTrp 1 5 AGC CCA ACA ACC TAT CTC CTC CTG CTG CTG CTG CTG AGC TCG GGA CTC101 Ser Pro Thr Thr Tyr Leu Leu Leu Leu Leu Leu Leu Ser Ser Gly Leu 1015 20 AGT GGG ACC CAG GAC TGC TCC TTC CAA CAC AGC CCC ATC TCC TCC GAC149 Ser Gly Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp 2530 35 40 TTC GCT GTC AAA ATC CGT GAG CTG TCT GAC TAC CTG CTT CAA GAT TAC197 Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr 4550 55 CCA GTC ACC GTG GCC TCC AAC CTG CAG GAC GAG GAG CTC TGC GGG GGC245 Pro Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly 6065 70 CTC TGG CGG CTG GTC CTG GCA CAG CGC TGG ATG GAG CGG CTC AAG ACT293 Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr 7580 85 GTC GCT GGG TCC AAG ATG CAA GGC TTG CTG GAG CGC GTG AAC ACG GAG341 Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu 9095 100 ATA CAC TTT GTC ACC AAA TGT GCC TTT CAG CCC CCC CCC AGC TGT CTT389 Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu 105110 115 120 CGC TTC GTC CAG ACC AAC ATC TCC CGC CTC CTG CAG GAG ACC TCCGAG 437 Arg Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu125 130 135 CAG CTG GTG GCG CTG AAG CCC TGG ATC ACT CGC CAG AAC TTC TCCCGG 485 Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg140 145 150 TGC CTG GAG CTG CAG TGT CAG CCC GAC TCC TCA ACC CTG CCA CCCCCA 533 Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Pro Pro Pro155 160 165 TGG AGT CCC CGG CCC CTG GAG GCC ACA GCC CCG ACA GCC CCG CAGCCC 581 Trp Ser Pro Arg Pro Leu Glu Ala Thr Ala Pro Thr Ala Pro Gln Pro170 175 180 CCT CTG CTC CTC CTA CTG CTG CTG CCC GTG GGC CTC CTG CTG CTGGCC 629 Pro Leu Leu Leu Leu Leu Leu Leu Pro Val Gly Leu Leu Leu Leu Ala185 190 195 200 GCT GCC TGG TGC CTG CAC TGG CAG AGG ACG CGG CGG AGG ACACCC CGC 677 Ala Ala Trp Cys Leu His Trp Gln Arg Thr Arg Arg Arg Thr ProArg 205 210 215 CCT GGG GAG CAG GTG CCC CCC GTC CCC AGT CCC CAG GAC CTGCTG CTT 725 Pro Gly Glu Gln Val Pro Pro Val Pro Ser Pro Gln Asp Leu LeuLeu 220 225 230 GTG GAG CAC TGACCTGGCC AAGGCCTCAT CCTGCGGAGC CTTAAACAAC774 Val Glu His 235 GCAGTGAGAC AGACATCTAT CATCCCATTT TACAGGGGAGGATACTGAGG CACACAGAGG 834 GGAGTCACCA GCCAGAGGAT GTATAGCCTG GACACAGAGGAAGTTGGCTA GAGGCCGGTC 894 CCTTCCTTGG GCCCCTCTCA TTCCCTCCCC AGAATGGAGGCAACGCCAGA ATCCAGCACC 954 GGCCCCATTT ACCCAACTCT GAACAAAGCC CCCG 988 235amino acids amino acid linear protein 6 Met Thr Val Leu Ala Pro Ala TrpSer Pro Thr Thr Tyr Leu Leu Leu 1 5 10 15 Leu Leu Leu Leu Ser Ser GlyLeu Ser Gly Thr Gln Asp Cys Ser Phe 20 25 30 Gln His Ser Pro Ile Ser SerAsp Phe Ala Val Lys Ile Arg Glu Leu 35 40 45 Ser Asp Tyr Leu Leu Gln AspTyr Pro Val Thr Val Ala Ser Asn Leu 50 55 60 Gln Asp Glu Glu Leu Cys GlyGly Leu Trp Arg Leu Val Leu Ala Gln 65 70 75 80 Arg Trp Met Glu Arg LeuLys Thr Val Ala Gly Ser Lys Met Gln Gly 85 90 95 Leu Leu Glu Arg Val AsnThr Glu Ile His Phe Val Thr Lys Cys Ala 100 105 110 Phe Gln Pro Pro ProSer Cys Leu Arg Phe Val Gln Thr Asn Ile Ser 115 120 125 Arg Leu Leu GlnGlu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp 130 135 140 Ile Thr ArgGln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro 145 150 155 160 AspSer Ser Thr Leu Pro Pro Pro Trp Ser Pro Arg Pro Leu Glu Ala 165 170 175Thr Ala Pro Thr Ala Pro Gln Pro Pro Leu Leu Leu Leu Leu Leu Leu 180 185190 Pro Val Gly Leu Leu Leu Leu Ala Ala Ala Trp Cys Leu His Trp Gln 195200 205 Arg Thr Arg Arg Arg Thr Pro Arg Pro Gly Glu Gln Val Pro Pro Val210 215 220 Pro Ser Pro Gln Asp Leu Leu Leu Val Glu His 225 230 235 71base pairs nucleic acid single linear cDNA to mRNA NO NO 7 AATTGGTACCTTTGGATAAA AGAGACTACA AGGACGACGA TGACAAGACA CCTGACTGTT 60 ACTTCAGCCA C71 37 base pairs nucleic acid single linear cDNA to mRNA NO NO 8ATATGGATCC CTACTGCCTG GGCCGAGGCT CTGGGAG 37

What is claimed is:
 36. A hematopoietic cell expansion media comprisingcell growth media, and flt3-L alone or in combination with a cellulargrowth factor, wherein the flt3-L and the growth factor are in an amountsufficient to cause hematopoietic cell expansion.
 49. A method forexpanding hematopoietic cells comprising contacting the cells withflt3-L alone or in combination with a cellular growth factor, whereinthe flt3-L and the growth factor are in an amount sufficient to causehematopoietic cell expansion.